Light microscopic imaging of living cells

Critical parameters:

• Low light level

• Speed of data acquisition

• Quantitative measurements

• “in vivo” environment

Fluorescence / imaging microscope

Xe arc lamp

Intensified video camera

Filter Wheel

Dichroic mirror

Video frame grabber

I. Optics and image formation

Video monitor

Fluorescence / imaging microscope

Thermostatized perfusion chamber

Manifold

Perfusion reservoirs

II. Handling / observation of living cells

Metafluor® Software: Fluorescence ratio imaging system

• Image acquisition via video frame grabber

• Image processing, analysis and quantification

• Real time processing (averaging, background

subtraction, ratio images)

• Automation: Control of external devices (filter

wheels, monochromators, valves, triggers,

etc.)

• Implemented on Pentium II 333 MHz computer

Some fluorescent dyes that can be used:

Fluorescein-, Rhodamine-based dyes

pH: BCECF, SNARF

Ca2+: Fura-2, Fluo-3, Mag-Fura-2, Calcium Green, Rhod-2

Na+: SBFI, SBFO, Sodium Green

K+: PBFI, CD 222

mitochondrial : Rhodamine 123, TMRE

Vesicle release: FM 1-43

Autofluorescence: NAD(P)H

Green Fluorescent Protein (GFP)

etc.

FUTURE ACQUISITIONS

UV Laser for flash photolysis experiments

Motorized focus control for Nikon microscope

Digital deblurring software (e.g. EPR (Scananlytics) or

Autodeblur (AutoQuant)

Liquid crystal tunable filter (LCTF) for spectral analysis of

emission

AVAILABILITY

On collaboration basis

Contact person: J.-Y. Chatton (Phone 692-5575)

Na+ homeostasis in astrocytes (simplified model)

3 Na+1 Glut

1 K+

1 H+

Na+

K+

ATP

[Na+]o=160 mM

[Na+]i10 mM

Na+ AKR

GlutKainateAMPA

Structure of SBFI (sodium-binding benzofuran isophthalate)

380nm-Image Control (ratio image)

L-Glu 1mM(ratio image)

Glutamate-evoked intracellular Na+ changes

0

10

20

30

40

50

60

[Na

+] i

(mM

)

L-Glu 10M 100M 200M 1000M

5 min

201098/Exp3

Glutamate: concentration-response analysis

0

20

40

60

80

100

120

0.01 0.1 1 10 100 1000

[Na+

] i am

plitu

de (

% m

ax. r

espo

nse)

Glutamate (M)

Km = 6.78 ± 1.7 M

Na+ homeostasis in astrocytes (simplified model)

3 Na+1 Glut

1 K+

1 H+

Na+

K+

ATP

[Na+]o=160 mM

[Na+]i10 mM

Na+ AKR

GlutKainateAMPA

Effect of Na+/K+-ATPase inhibition (1)

0

10

20

30

40

50

21.01.99 - Exp 1a

[Na+

] i (

mM

)

4 min

Glutamate

Ouabain

Jean-Yves Chatton / P.J. Magistretti (Physiologie, Lab 204 Annexe)

Primary applications: dynamic changes of Ca2+i, pHi , Na+

i , etc. inliving cells

EQUIPMENT

Nikon inverted microscope (Diaphot 300)

Xe-lamp with filter wheel (Lambda 10-2, Sutter Instr.)

Intensified CCD camera Gen III+ intensifier (ICCD 350F

VideoScope)

Video frame grabber Tech MV-1000

Pentium II 333 MHz computer (Windows 95)

Image archiving: CD-R/W Yamaha (4x2x6) (Software:

WinOnCD)

Fast perfusion chamber w/ temperature control (on-site

development)

SOFTWARE

Metafluor v. 3.5 (Universal Imaging) for time-lapse acquisition

and quantitative analysis of fluorescence images

Lucia M v. 3.51 (LIM) ) acquisition and analysis of fluorescence

images (general-purpose application)

Scion Image (v. Beta 3) installed on Win95 and on Win NT4.0

machines.

Microtome (Scanalytics, DOS version with ISA Processing

board) older version – not yet installed