light microscopic imaging of living cells critical parameters: low light level speed of data...
TRANSCRIPT
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