Basic Quantitative MicroscopyPractical Pitfalls in Image Acquisition

Johanna Dela Cruz2 March 2016

What are my imaging goals?

• resolution• contrast• remove the greatest amount of out-of-focus light• maintain a detectable signal• minimize cell phototoxicity• minimize fluorophore bleaching• speed• multidimensional• image area

How should I image my sample?

• Brightfield illumination (BF)• Phase contrast (PC)• Differential interference contrast (DIC)• Darkfield (DF)• Polarized light

Transmitted light microscopy

• Standard Widefield• Confocal/multiphoton/TIRF/PALM/STORM/

STED

Incident or reflected light/epi-

illumination

• Brightfield illumination (BF)• Phase contrast (PC)• Differential interference contrast (DIC)• Darkfield (DF)• Polarized light

Transmitted light microscopy:

Stained/Unstained samples

Halogen lamp

condenser

Specimen

objective

ocular

• Brightfield illumination (BF)• Phase contrast (PC)• Differential interference contrast (DIC)• Darkfield (DF)• Polarized light

Incident or reflected light/epi-

illumination: Metals; opague;

Fluorescence-labeled

Halogen lamp

condenser

Specimen

objective

ocularMercury lamp

Dichroic Filter

• Brightfield illumination (BF)• Phase contrast (PC)• Differential interference contrast (DIC)• Darkfield (DF)• Polarized light

Transmitted light microscopy:

Stained/Unstained samples

• Standard Widefield fluorescence• Confocal/multiphoton/TIRF/super-

resolution

Incident or reflected light/epi-illumination:

Metals; opague; Fluorescence-labeled

Brightfield Brightfield Darkfield

BF: contrast generated from changes in light absorption, refractive index or color

DF: scattered light caused by optical discontinuities

Phase Contrast DIC FluorescencePC: contrast from

interference of light path lengths through sample

DIC: use of light-shearing prisms and polarized light to exaggerate minute differences in specimen

thickness gradients and refractive index

• Thin samples

• Optical sectioning of thick samples

Conventional Widefield or Confocal?

• Thin samples

• Optical sectioning of thick samples

Conventional Widefield or Confocal?

Widefield fluorescence microscopy

Confocal microscopy(optical slice projection)

fluorophore is excited with one wavelength of light and light of a different wavelength is

emitted

Fluorescence

Upright vs. Inverted Microscopes

- No fundamental difference in the ability to produce and channel light along various paths

- Image quality dependent on sample preparation, objective lenses, light source and wavelength, fluorophore filter set, detector

Sample Preparation“Garbage in = Garbage out”

• Non-fluorescent contrasting agents/stains – immunohistochemical• Fluorophores: extinction coefficient, quantum yield, photostability• fixed-cell imaging: fixation and permeabilization, signal enhancers• Immunofluorescence imaging: antibodies• Live-cell imaging: imaging media, background suppressors• Type of mountant used:

PVA vs glycerol-based Anti-fade agents and compatibility with fluorophores Coverslip sealant (nail polish, VaLaP)

Optical properties of the microscope that you need to know about

Objective lens – most critical component of a microscope• Magnification• Numerical aperture (NA): light-gathering ability

- resolving power- signal intensity/brightness ∞ (NA)4 / Mag2

Which would you choose?40x, 0.75 NA vs 40x, 1.3 NA60x,1.4 NA vs 100x, 1.4 NA

Manufacturer

Class/Special designation

Magnification

Numerical aperture, NA

Immersion medium

Specialized optical properties/ contrast method

Infinity-correctedCoverslip thickness

Color of objective text: contrast methodstandardpolarization/DICphase

Color coding of Immersion fluidoiloil/water/glyceringlycerin (glyc)water (W)

Color coding of Magnification4x/5x10x20x40x63x100x/150x

Correction collar• Cover glass thickness

correction• Different immersion• Different temperature

Coverglass thickness• 0.17: standard (#1.5)• 0 : w/o coverglass• – : insensitive

Resolution

• most important factor that determines image quality

• amount of detail you can see in an image

• Without a sufficiently high resolution, magnification is not possible without loss of quality.

Finest observable details ~ λ/2NA

Factors Affecting Resolution

• Objective-related: correction for aberration, NA• Specimen-related: coverglass, mounting medium, immersion

medium• Lighting: excitation wavelength, color range• System stability

Numerical Aperture

Olympus Primerhttp://olympus.magnet.fsu.edu/primer/java/nuaperture/index.html

NA = n sin(θ)n = refractive index

(1.0 -> air)Θ = angular aperture

0.25 NA

0.5 NA

0.95 NA

Numerical Aperture

40x/0.6 40x/1.3

Other features of the objective may prove more critical for a particular sample or application:

Why would anybody choose an objective of lower NA?

• Working distance: how far objective can focus into sample

• Design for use with/without coverslips

• Corrections for flatness of field, chromatic and spherical aberrations

• Transmission of specific wavelengths (UV, IR)

• Refractive index of immersion medium proportional to NAoil water air (decreasing NA)

• Brightness: which would you choose? 40x/1.2 W vs 63x/1.4 oil

major cause of the loss in signal intensity and resolution with increasing focus depth through thick specimens

Spherical Aberration

Increased by:• use of the wrong coverslip thickness• type of immersion oil • thick layer of mounting medium• presence of air bubbles in the immersion or

mounting medium• temperature change use of oil immersion on aqueous sample

L: at 0 um; R: at 35 um into sample

wavelength-dependent artifacts that occur because the refractive

index of every optical glass formulation varies with

wavelength

Chromatic Aberration

Factors Affecting Resolution

• Objective-related: correction for aberration, NA• Specimen-related: coverglass, mounting medium, immersion

medium• Lighting: excitation wavelength, color range• System stability

• Coverglass: protect specimen integrity and provide a clear window for observation

• Introduces chromatic and spherical aberration (loss of contrast) that must be corrected by objective lens

• Thickness: 0.13 to 0.22 mm; standard: 0.17 mm• Negligible for dry objectives with NA < 0.4; significant at NA > 0.65

• Effect of mounting medium

Coverglass and Mounting Medium

Matching Immersion Medium to Objective

• Immersion media: used to minimize the refractive index (RI) differences present in space between objective and sample includes substrate (glass coverslip) sample is on and imaging

medium (buffer) sample is in.

• NA partly depends on the RI of the immersion medium [NA = n sin(θ)]

• Consequences of Mismatched RIs: spherical and chromatic aberrations, loss of resolution, reduced scan depth in z, wasted time and effort

Factors Affecting Resolution

• Objective-related: correction for aberration, NA• Specimen-related: coverglass, mounting medium, immersion

medium• Lighting: excitation wavelength, color range• System stability

• important factor in microscope resolution

• Resolution limited to ~1/2 wavelength of illumination

• Shorter wavelengths (near UV) yield higher resolution

Wavelength

Know your fluorophores, excitation sources and filter sets

Excitation spectrum

Emission spectrum (fluorescence)

Know your fluorophores, excitation sources and filter sets

• Emission wavelength is independent of the excitation wavelength• emission intensity is proportional to the amplitude of the excitation wavelength,

Mercury arc light output

Know your fluorophores, excitation sources and filter sets

• Emission wavelength is independent of the excitation wavelength• emission intensity is proportional to the amplitude of the excitation wavelength,

Laser output at 405 nm

Multiple labeling

Choosing fluorochromes with well-separated excitation and emission spectra is critical

Emission Filters

Band-pass filters collect emissions in a specific range. The narrower the range of the band-pass filter, the better it can separate fluorochromes with close emission spectra.

Simultaneous vs Sequential Imaging

Simultaneous Acquisition: bleedthrough/cross-talk

Sequential Acquisition: well-separated

• Start at the lowest laser power possible, with a higher PMT voltage (>600 V) and gradually increase the laser power as required

• Averaging: better signal-to-noise ratio

• More light in plane of focus does not increase signal intensity: more out-of-focus fluorophores excited can lead to poorer z-axis resolution and increased photobleaching/phototoxicity

Fluorophore Signal

Sampling Frequency: Undersampling and Oversampling

Undersampling Good sampling Oversampling

Large pixels – low resolutionBrighter, less exposure,

less photobleaching

Too small pixels – excess of information,

dimmer, increased bleaching, waste of time & storage

Nyquist sampling

• CCD camera: averaging or binning increases S/N but decreases resolution• Binning: useful for large data sets or live-cell imaging, where binning and

shorter exposure times can be used to reduce phototoxicity• Binned images can be blurry: undersampling• using high NA objectives, making pixels smaller: does not always add

resolution because objects that are smaller than the wavelength of light cannot be resolved

• high zoom: smaller pixels, more data points, larger image files --specimen is oversampled, additional structural information not attained. For visible light and high NA objectives (>0.8) a pixel size of ∼0.1-0.2 μm is ideal.

Offsets and Detector

Saturation:

Avoid data clipping

Offset too high

Gain too high

Software Settings and Image Display

Original image Brightness/contrast enhanced

Gamma adjusted

• use the full dynamic range of the detector• Image display settings• best to display images in grey scale whenever possible• Dim features can be enhanced by modifying the display to a non-linear

LUT using the gamma factor • Display settings do not change the underlying data, however, these

manipulations should be mentioned in figure captions

Software Settings and Image Display

• The most critical components of the fluorescence microscope for quantitative imaging: objective lens, emission filter, and detector

• Wide-field microscopy: often inappropriate for quantitation because you collect emitted light from the whole sample depth without knowing the thickness of each cell or structure

• Confocal microscopy: more quantification-friendly for samples > 15–20 um in depth due to defined optical section thickness

• Deeper focal planes will show reduced signal intensity due to absorption and scatter, necessitating further, more complex corrections

Quantification of images—why is it useful and when is it appropriate?