ADVANCED METHODS IN BIOENGINEERING … 1 ADVANCED METHODS IN BIOENGINEERING LABORATORY • Introduce...

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ADVANCED METHODS IN BIOENGINEERING LABORATORY

• Introduce professors

• Content & structure

• Safety

• Logistics

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Introduce Professors

Georg Fantner LBNI ‐ Laboratory for Bio‐ and Nano‐ Instrumentation

Lab website, lbni.epfl.ch [email protected]

Carlotta Guiducci CLSE ‐ Chair on Engineering. Laboratory of Life Sciences Electronics.

Lab Website: clse.epfl.ch [email protected]

Aleksandra Radenovic LBEN ‐ Laboratory of Nanoscale Biology LBEN

Lab website: lben.epfl.ch [email protected]

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Objectives1. Learning the quantitative approach in bioengineering

How to make use of quantitative high‐sensitive, high resolution technologies to measure biophysical and biochemical parameters

Imaging, trapping and tracking single biological entities at the nanoscale

Characterizing molecular binding by the interpretation of averaged signal on millimeter‐square areas

Determining sensitivity and selectivity of a biosensor

Designing and building a lab‐on‐a‐chip device (and some of the things you can do with it)

How to analyze real life scientific data (get quantitative answers)

2. working in real‐life research labs Keeping professional notebook

Behavior in a cleanroom

3. Planning, organizing and executing a research project

4. Write a scientific paper in the “Letter to nature” style

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CONTENT AND STRUCTURE

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Teaching method/Structure of the course

4 contact hours per week, 4 credits

One ex‐cathedra introduction session (1 week)

3 modules (6 weeks) of pre‐prepared exercises

One “independent” research project based on a real world publication/project (1 week planning + 3 weeks execution)

Write a paper in “Nature style” about your project (2 weeks)

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Schedule

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Schedule ABML 2016‐2017

GR1 GR2 GR3 GR4 GR5 GR6

1st 02/20 INTRO INTRO INTRO INTRO INTRO INTRO HOME: prepare for experiments on handouts

2nd 02/27 SPR AFM OT LOAC SD BM HOME: analyse experiments and prepare for experiments on handouts

3rd 03/06 SPR AFM OT LOAC SD BM HOME: analyse experiments and prepare for experiments on handouts

4th 03/13 SD SPR LOAC OT BM AFM HOME: analyse experiments and prepare for experiments on handouts

5th 03/20 SD SPR LOAC OT BM AFM HOME: analyse experiments and prepare for experiments on handouts

6th 03/27 OT SD AFM BM SPR LOACPreference of exercise sent by students HOME: analyse experiments and prepare for experiments on handouts

7th 04/03 OT SD AFM BM SPR LOAC

Assignement of projects communicated by teachers HOME: analyse experiments and prepare plan

8th 04/10

Meeting with teachers and brainstorming on plan of experiments HOME: work on plan

9th 04/24 EXP HOME: work on analysing experiments and on paper



12th 05/15 EXPhand first version of the paper by Friday HOME: work on analysing experiments and on paper

13th 05/22

feedback from teacher and assistants on the first version of the paper HOME: work on finalizing paper

14th 05/29

work with assistants on paper

hand final version of paper by Friday end of the semester HOME: work on finalizing paper

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Groups

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GR1 Chansin Jonathan Bryan Maoha

Clément Blandine Françoise

GR2 Cuttaz Estelle Annick

Durtschi Moritz

GR3 Hofer Moritz

Holmvik Christian

GR4 Martin Octave François Georges

Pagano Veronica

GR5 Soukouti Line

Sverrisson Freyr

GR6 Wannebroucq Quentin Olivier Pierre

Teaching method/Structure of the work Exercises consist of four hours of supervised work in group. Independent work (with available support from the teacher

and assistants during specified office hours): Learn content presented in the introductory lecture Prepare each exercise prior to the first session on the available

handouts and complementary material. Complete of data analysis when requested after the end of the

exercise Laboratory Notebook‐filling during and after (for analysis only,

not for rewriting) the practice.

Independent research project, with support from assistant and teachers.

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Teaching material

The handouts, applets and additional material for respective exercise can be found on our teaching website http://lben.epfl.ch/teaching

NO MOODLE!!! Reference books:

Intermolecular and Surface Forces, J. Israelachvili, Academic press Surface Plasmon resonance Based Sensors, J.Homola et al., Springer Surface Design: Applications in Bioscience and Nanotechnology, R. Forch, H.

Schonherr, A.T. Jenkins, Wiley "Introduction to Error Analysis: The Study of Uncertainties in Physical

Measurements," Taylor, John R., 1997, University Science Books, Optical Trapping Review : K.C. Neuman & S.M. Block, "Optical trapping,”Rev.

Sci. Instrum. 75 (2003). Lab on a Chip Technology, Volume 1: Fabrication and Microfluidics, Keith E.

Herold and Avraham Rasooly, Caister Academic Press, 2009 http://www.afmworkshop.com/atomic‐force‐microscope‐book.php

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Evaluation

Continuous control: 2/3 Paper written during independent research project

1/3 Evaluation of the quality of the lab notebook, and evaluation by the TAs (quiz)

• compile properly the lab notebook (one for each student)

• Prepare the exercise in advance by studying the handouts provided on the site

• You will have to study the handouts (provided on the web site) and prepare for the exercises beforehand. At the beginning of each exercise, a quiz proposed by TA will serve to assess your preparation. If failed, you will get a ZERO POINTS for this part

• Participate actively and as much as possible autonomously to the exercise

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THE SIX LABORATORY PRACTICES

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Bioanalytics SURFACE DESIGN The students will learn some basic techniques of surface design for

bioanalytics.

SURFACE PLASMON RESONANCE The students will learn how to plan and interpret surface bio‐molecular binding experiments.

Working with single biological entities BROWNIAN MOTION The students will learn how to simulate and analyze Brownian motion

of single particles in Matlab, use brightfield and darkfield microscopy. They will be introduced to the image data acquisition, theory and software design for image filtering and particle tracking in Matlab.

OPTICAL TRAPPING In this students will learn the basics of operating a high‐end optical tweezers to record mechanical transitions of single molecules.

ATOMIC FORCE MICROSCOPY The students will learn how to use AFM on various biological samples. Learn image processing and how to extract meaningful data at the nanometer scale.

LAB‐ON‐A‐CHIP The students will learn how to design and fabricate miniature chemical and bio‐chemical

analysis systems, also known as Lab‐on‐a‐Chip systems, referring to the idea of shrinking a complete chemical analysis laboratory onto a small chip.

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AFM a Versatile Tool for NanoscaleMeasurements

Things you will learn: Make surface topography images with sub

nanometer resolution Nanoscale imaging of E.coli. Extract tendons from rat tails, image

microfibrils of collagen and measure the characteristic D‐banding structure of collagen fibrils

How to process and analyze AFM data

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Locations and dress code

AFM dress code: wear pants and close toed shoes. Bring your lab coat.

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SPR

Description:

The exercise consists in the employment of label‐free biosensors for the observation of binding kinetics. Real‐time biomolecular bindings will be observed for different molecules.

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Objectives: understanding the importance of real‐time measurements of biomolecular

binding interactions Perform kinetic analysis for ligands immobilized on a sensor chip by amine

coupling chemistry Direct coupling Ligand‐mediated coupling

Structure: 1st week: Introduction to SPR Technology and Surface preparation 2nd week: SPR experiment and kinetic analysis


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SURFACE PLASMON RESONANCE location: TP SSV and CLSE BM2112 Meeting in TPSSV for first session and in front of BM2112 for second. dress code: lab coat. Closed shoes. Long pants.

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Surface designDescription:Modern bioanalytics is based on surface detection of biomolecules. The exercise will explore a surface modification technique commonly employed in biosensor and microbiosensors.

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Objectives:Learn how to design an experiment of biomolecular detection on arrayed surfacesPerform an analysis in terms of hybridization efficiency according to different conditionsStructure:1st week: surface cleaning and deposition of an arrayed pattern of molecular probes (DNA oligonucleotides)2nd week: hybridization with complementary sequence, data acquisition and analysis


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SURFACE DESIGN location: TP SSV

dress code: lab coat. Closed shoes. Long pants

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Brownian motion

In the first part of this exercise, the students will replicate Perrin's work with modern equipment. Next they will investigate intracellular vesicle transport inside living cells and determine if the vesicle transport is accomplished by Brownian motion or by directed transport

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Aleksandra Radenovic


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BROWNIAN MOTION location: MED 3 1159.

dress code: lab coat

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Optical Trapping

Optical trapping is one of the most successful technology transfers from a physics lab to biology. The goal of this exercise is to provide hands on experience to the bioengineering

students of one of the mostly used single molecule technique.

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OPTICAL TRAPPING location: MED 2 1117

dress code: lab coat, IR goggles

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Lab‐on‐a‐chip

Lab‐on‐a‐chip (LOC) exercise will introduce students to the fundamental elements of moving fluids in LOC systems such as flows, pressure driven flow, electro‐osmotic driven flow, capillary effects, surface forces.

AMBL 2016/2017 23Aleksandra Radenovic

Locations and dress code LAB‐ON‐A‐CHIP

location:• CMI+ zone 12. dress code: wear pants. You will be given instruction on what to dress‐LOC • LBEN lab BM2124

meeting for the first session in front of CMI+ and for the second session in BM5202

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CMI+

LBEN

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ANALYSIS SESSIONS Location: BM5202 (for AFM, Brownian motion and Optical trapping exercises)

LAB SAFETY

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Safety

In any laboratory, there is potential for injury if certain common‐sense practices are not followed. In AMBL this is minimal, but it’s still important to follow a few basic rules.

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Electrical Safety

Electrical injuries happen when large amounts of electrical power are dissipated by the body. Most often, this happens in high‐current situations, which is why you always hear that “it’s not the voltage, it’s the current that is dangerous.” Strictly speaking, both are dangerous, and it’s a good idea to avoid becoming a current path.

In AMBL, we will work with only low‐power electronics, and nothing we do is likely to cause injury. However, some common‐sense precautions, are in order:

– don’t connect supply voltages directly to ground – don’t touch any current‐carrying conductor with your bare hands

These simple rules will keep you from injuring yourself and damaging circuit components. Some components will have maximum power ratings that should not be exceeded, so pay attention to these values.

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Chemical Safety & Biosafety

Though there is minimal wet work in AMBL please do not bring food or drink into the lab. The electronics will appreciate it, and we will also later be handling some bacteria and fluorescent dyes.

When needed, latex gloves will be provided, as well as proper containers for disposing of chemical/biological waste and sharps. Please make sure to wash your hands with soap and water after removing gloves and before leaving the lab. Please report any spills or injuries to the lab instructor immediately

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HOW TO KEEP A GOOD LAB NOTEBOOK

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Why is it Important to Keep a Good Laboratory Notebook?

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Keeping a complete and accurate record of experimental methods and data is a vital part of science and engineering. Your laboratory notebook is a permanent record of what you did and what you observed in the laboratory. Learning to keep a good notebook now will establish good habits that will serve you throughout your career. Your notebook should be like a diary, recording what you do, and why you did it.

You should feel free to record your mistakes and difficulties performing the experiment ‐ you will frequently learn more from these failures, and your attempts to correct them, than from an experiment that works perfectly the first time. It is extremely important that your notebook accurately record everything you did. A good test of your work is the following question: could someone else, with an equivalent technical background to your own, use your notebook to repeat your work, and obtain the same results?

For that matter, could you come back six months later, read your notes, and make sense of them? If you can answer yes to these two questions, you are keeping a good notebook. Aleksandra Radenovic

Why is it Important to Keep a Good Laboratory Notebook?

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The laboratory notebook forms a permanent record that can be referred to while completing a disclosure report (often the first step in patent preparation) and later, provides accurate documentation of the work done. When an investigator makes an invention during the course of a research project, the dates of the conception and reduction to practice (turning an idea into a reality) become very important. Generally, a sketch and a brief written description are sufficient to establish conception. Reduction to practice is accomplished by actually constructing and successfully testing a material or device incorporating the invention.

During prosecution of a patent application before the U.S. Patent Office, or even after issuance of a patent, the filing of another patent application may initiate an interference proceeding to determine which party was the first to invent. Each party has an opportunity to submit documentary proof of his or her dates of conception and reduction to practice. A laboratory notebook may be, and in several high‐profile cases has been the crucial piece of evidence in this procedure.


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What your notebook is for

Your lab notebook serves three important purposes:

1. A record of important procedures for experiments you have developed during your experimentation.

1. A record of the results of experiments that you have performed.

1. The means to reproduce the results of your experiments by following the procedures you have developed at another time or place.

A good notebook is not simply a list of results of experiments but allows you to develop methods that you can use for further experimentation and would allow someone else to reproduce your results and understand why you did what you did in your experiments.

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What goes into your notebook

Page numbers – if your notebook doesn’t already have them add them to the upper outside corner of each page. These are important so you can refer back to frequently used tables, procedures, or results. You can also be sure that there are no missing pages (leading to missing steps) if following a past procedure.

A table of contents – The first few pages should be reserved for this, it allows you to quickly find the information you are looking for and makes the book a useful reference. Later on you will be able to find a particular experiment without having to read every page.

Dates – Every entry, or at the very least every day that you record data should be dated, this allows you to more easily.

Unusual conditions during an experiment – Sometimes things go differently than we plan and we have something unusual happen during our experiments some of the things you might want to look for and record are: Strong storms (ie. behavior of an observed animal may be atypical) Extremes in temperature or humidity (many instruments and materials are sensitive to temperature and humidity) Power failures (if your experiment requires power)

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What goes into your notebook (cont.ed)

Name of the corresponding files.

Something is went wrong or was unexpected (ie. you notice that the apparatus is no longer working at some point during your experiment)

Experimenter fatigue may impair your ability to make good observations

Reasons for decisions made during an experiment – What we did isn’t always good enough, why we did what we did is just as important to record. Make sure that you record the whys and not just the whats. Contact information for people that provided you with information or supplies – They may be able to provide you with some materials in the future or to give you more information later on should you need it. It is important to give credit where it is due as well. Any information that you might need to reproduce the results of an experiment – Your notebook alone should be sufficient for someone to reproduce your experiment. Aim to be as complete as possible!

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Rules for Maintaining your Laboratory Notebook

Leave several pages blank at the beginning for a Table of Contents and update it when you start each new experiment or topic

Always use pen and write neatly and clearly

Date every page on the top outside corner

Start each new topic (experiment, notes, calculation, etc.) on a right‐side (odd numbered) page

Record the TITLE and OBJECTIVES of each experiment (or notes or calculations) at the top of the first page of the notebookdedicated to this topic.

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Rules for Maintaining your Laboratory Notebook

If you make a mistake, don’t obliterate it! You may need to read your mistake later – perhaps you were right the first time! Use a single cross out and EXPLAIN why it was an error.

Data typed into the computer must be printed and taped into your lab notebook. Plots of data made in lab should also be printed and taped in your lab notebook.

When you record an observation in your notebook, include an explanation of what you were doing at the time. If appropriate, youmay just record the step number in the instructions followed by your observation

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Example: Complete Experiment

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Key points in this example:1. Neat and legible handwriting2. Experiment title and purpose clearly stated3. Procedure described clearly and succinctly, including

errors and the steps taken to correctthem4. Computations performed neatly showing intermediate

steps5. Errors crossed out with a single line and explained6. All pages dated at the top and signed by lab professor

on the same

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INTRODUCTION TO ERROR ANALYSIS

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No measurement made is ever exact!

The accuracy (correctness) and precision (number of significant figures) of a measurement are always limited by the degree of refinement of the apparatus used, by the skill of the observer, and by the basic physics in the experiment.

In doing experiments we are trying to determine the values for certain quantities, or trying to validate a theory.

We will give a range of possible true values based on our limited number of measurements.

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Random errors: Analog Instruments

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•Least count= 2.5sLeast Count: the smallest division or digit marked on an instrument

•Instrument limit of error≈ 1sInstrument Limit of Error (ILE): the precision to which a measuring device can be read, and is always equal to or smaller than the least count.

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Digital Instruments

In this case: Instrument limit of error (ILE)= Least count

BEWARE: Not all digits are full digits. (half digits can be either 0 or 1, usually on the left)

BEWARE: Not all digits are meaningful

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Significant Digits

A significant figure is any digit 1 to 9 and any zero which is not a place holder.

Examples:

1.350 there are 4 significant figures

0.00320 there are 3 significant figures

1350 there are ??? Significant figure

use scientific notation: 1.35×103 has 3 significant digits

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How many significant figures should be in the final answer?

In running computations we maintain numbers to many figures, but we must report the answer ONLY to the proper number of significant figures.

The short rule for multiplication and division is that the answer will contain a number of significant figures equal to the number of significant figures in the entering number having the least number of significant figures.

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Estimated Uncertainty by Repeated Measurements

repeat the measurement several times, find the average, and find either the average deviation or the standard deviation.

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Average deviation Standard deviationAverage

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Example:

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We can report the answer as (15.5 ± 0.1)m for average deviation and (15.5 ± 0.2)m for standard deviation

How to calculate the standard deviation?1. Compute the square of the difference between each value and

the sample mean.

2. Add those values up.

3. Divide the sum by n‐1. This is called the variance.

4. Take the square root to obtain the Standard Deviation (sample standard deviation: Bessel’s correction).

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Why make many measurements? Standard Error of the Mean.

The standard deviation (SD) is how spread out THINGS in the population are, and this is calculated (somehow) from the data in your sample. It is useful in describing the population itself.

The standard error (SE) is how spread out the SAMPLE MEAN will be around the true population mean. It is useful in describing how close your results will be to the right answer.

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When to use SD and when SE

Decide if we are measuring one value multiple times (use standard error), or if we are measuring one quantity in multiple cases (use standard deviation).

Another way to decide if you imagine you could make a perfect measurement, would you always get the same number, then use the standard error.

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Examples

When we describe a population of cells, by measuring their length, we will calculate the mean and the standard deviation, because there is no ”right length”.

When we want to measure the weight of a sample, we will calculate the average weight and the standard error, because there is a ”right weight”.

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How are Standard Error and Standard Deviation Related?

The standard error of the mean in the simplest case is defined as the standard deviation divided by the square root of the number of measurements.

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Relative vs. Absolute Errors

absolute value of the error (absolute error), for example R = (33 ± 1.65)kΩ

Relative error: absolute error/value

error percentage R = (33 ± 5%)kΩ

These three types of errors are often used in different fields. Absolute errors are often used to depict measurement errors, whereas percentage errors are often used for tolerances of parts or components. The relative error is useful for error propagation.

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Rules for treating outliers:

Use critical judgment!

Think if there could be a reason that the outlier is a valid data point

Rule of thumb: outlier: if a value is more than 4 times the standard deviation from the mean (calculated from the other values, disregarding the potential outlier), then you might disregard the data point in your further analysis.

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Propagation of Errors

Given x=(x±Δx) and y=(y±Δy), what is z(x,y)

There are two ways to get an estimate for the error of z.

simplified version: the guiding principle in all cases is to consider the most pessimistic situation.

proper statistical treatment of error propagation: use the standard deviations to calculate the resulting uncertainty

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Addition and Subtraction

Example: w = (4.52±0.02)cm, x = (2.0±0.2)cm, y = (3.0±0.6)cm.

Find z = x+y−w and its uncertainty.

z = x + y − w = 2.0 + 3.0 − 4.5 = 0.5cm

For the simplified method we get: ∆z = ∆x + ∆y + ∆w = 0.2 + 0.6 + 0.02 = 0.82 rounding to 0.8 cm:

So z = (0.5 ± 0.8)cm

When using the standard deviation we get: ∆z = √(0.222 + 0.622 + 0.0222) = 0.633:

So z = (0.5 ± 0.6)cm.

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Multiplication by an exact number

When multiplying a measurement value with an exact number, multiply the uncertainty also with the exact number.

Example: The radius of a circle is r = (3.0 ± 0.2)cm. Find the circumference and its uncertainty

C=2πr = 18.850cm

∆C=2π∆r = 1.257cm (The factors of 2 and π are exact)

C=(18.8 ± 1.3)cm

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Multiplication and Division:

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Multiplication and Division with standard deviation

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Products of Powers z = xm + yn

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Mixtures of multiplication, division, addition, subtraction, and power

Treat the math of the uncertainties in the same order as you would treat the math itself!

Example:

w = (4.52±0.02)cm, x = (2.0±0.2)cm,

y = (3.0±0.6)cm.

Find z = wx + y2

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Mixtures of multiplication, division, addition, subtraction, and power

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An Answer without error estimates is meaningless!

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Safety and Behaviour in the cleanroom

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Description of the cleanroom

CMIBM+1

CMIBM‐1

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Air filtration and circulation

ACTUAL VALUES :(2/3 of maximum capacity)

• FRESH AIR- 38’000 m3/h- filter efficiency : 99.97% forparticles size 0.1-0.3 µm

• EXHAUST- 36 ’000 m3/h

• FFU- 167 units - 0.7 m2 active area- total: 189’000 m3/h- filter efficiency : 99.999% for particles size 0.1-0.3 µm

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Procedure to access CMI BM+1

• Quick access is possible to CMI BM+1

Contract to sign

Short project disscusion

Basic instruction are given orally

MSDS + SOP required if non standard chemical use

Attending the next full cleanroom safety training

• Enter via the “BM+1 SAS”,

lockers for personal items

cleanroom paper use only

• Transfer of materials and decontamination via “BM+1 SAS”

even small items must be decontaminated

no material enter without authorisation from CMI staff

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Dressing CMI BM‐1

•over shoes

•cleanroom suit

• ...

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Dressing CMI BM‐1

• over shoes

• cleanroom suit

• cleanroom boots

• face mask

• vinyl gloves

• safety googles

• CAMIPRO card

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Dressing CMI BM‐1 and BM+1

CMI BM ‐1 CMI BM +1

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Dressing CMI BM‐1 and BM+1

Get out the same way you get in

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General behaviour in the cleanroom

• NEVER WORK ALONE IN A ZONE

• NO MORE THAN 6 PEOPLE IN A ZONE

• WALK NORMALLY, DON’T RUN

• DO NOT SHAKE HANDS

• DO NOT WORK IN THE CLEANROOM IF YOU HAVE A COLD

• IN CASE OF EVACUATION ALARM, FOLLOW THE SAFETY RULES

• ONLY STAFF FIX THE MACHINES

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General cleanroom rules

• lint free cleanroom paper only

• cleanroom notebooks available through

CMi ordering system

• pens are available in each zone

• photocopier can be used to transfer notes

in and out of the cleanroom

• PC access to public folders in each zone

prohibited:• normal paper• pencils & normal pens

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CMI Website

http://cmi.epfl.ch/ Reservations

Orders

Safety and information

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Safety Rules

• Never Work Alone

• Only One Emergency N° :

• Report any safety problems you encounter

• Wear protective glasses or Medical glasses all the time

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Alarms & evacuation

• Double Tone Horn• Flashing Red Light Evacuate immediately with cleanroom dressing

meeting point : BM 1.125 (Ph. Flückiger office) wait there to be accounted for

remark : red alarm can be activated by the push-buttons

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Alarms & evacuation

Yellow alarm is for the staff only Eau DI / DI water

Neutralisation

…...

Single Tone HornFlashing Yellow light

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Alarms & evacuation

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Working Safely with Chemicals (1)

[1] NO NEW CHEMICALS without formal permission through SOP process

from Jean-Marie Voirol (CMi safety Manager)

[2] INFORM yourself

Read and understand Material Safety Data Sheets of all chemicals you intend to use, before using them

MSDS are available in the “SAS du Personnel”

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Working Safely with Chemicals (2)

[3] NEVER MIX chemicals (even with water)

Baths are always prepared by the CMi staff

Disposal of chemicals after use only in labelled containers

[4] NEVER STORE chemicals on working place

[5] RINSE and DRY working place after each use

before going to another equipment

[6] Use LABELLED CONTAINERS and tools for your processes

[7] If you have to leave, always label a chemical process in progress with name, date, your expected time of return, where you can be reached, the chemicals involved

[8] Never assume that colourless droplets are just water

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Laser safety

300 mW NIR diode lasers with λ=975nm (optical traps) The hazards of this Class IIIb laser come from its higher power level, and because it is invisible, making it harder to be aware of its location/direction. The beam will be largely constrained in the apparatus, and you will not need to make adjustments that might put you in the beam path. Safety goggles will be available, but not required.

In general, other important things to keep in mind:

Always know the path of the beam, and keep any body parts or reflective items (rings, watches, etc.) out of the beam path.

Always read the pre‐labs and know what special precautions you need to take associated with lasers or optics.

When in doubt about doing something, don’t do it before checking with the lab instructor.

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ADVANCED METHODS IN BIOENGINEERING … 1 ADVANCED METHODS IN BIOENGINEERING LABORATORY • Introduce...

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