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Laboratories for Molecular Medicine

Room 110, 110A, 111
70 Ship Street
Providence, Rhode Island 02903
Laboratories for Molecular Medicine

 

 

 

 

 

- Fluorescence Microscope
- Confocal Laser Scanning Microscopes
- Image Analysis

 

The Zeiss Axiovert 200M Fluorescence Microscope

Laboratories for Molecular Medicine, room 111

The Zeiss Axiovert 200M Fluorescence Microscope is a motorized inverted microscope with a Roper CoolSnap CF color camera and a Roper CoolSnap HQ monochrome camera controlled by MetaMorph 6.0 software.  This microscope is well equipped for transmitted light microscopy and fluorescence microscopy.

Objectives:

Table of Objectives
Mag
Correction NA
Phase
DIC
WD (mm)
Coverslip #
5x
Plan-NEOFLUAR 0.15
Ph1
-
13.6
0.17mm #1 1/2
10x
Plan-NEOFLUAR 0.3
Ph1
DIC II
5.5
0.17mm #1 1/2
20x
Plan-NEOFLUAR 0.5
Ph2
DIC II
2.0
0.17mm #1 1/2
40x
Plan-NEOFLUAR 0.75
Ph2
DIC II
0.5
0.17mm #1 1/2
40x Oil
Plan-NEOFLUAR 1.3
Ph3
DIC III
0.2
0.17mm #1 1/2
100x Oil
Plan-NEOFLUAR 1.3
Ph3
DIC III
0.2
0.17mm #1 1/2

 

Theoretical XY Objective Resolution: based on: Dxy=0.61*wavelength/NA

Mag
NA
DAPI
FITC
RHO
CY5
5x
0.15
1870 nm
2175 nm
2520 nm
2930 nm
10x
0.3
935 nm
1090 nm
1260 nm
1465 nm
20x
0.5
560 nm
650 nm
755 nm
880 nm
40x
0.75
375 nm
435 nm
505 nm
585 nm
40x Oil
1.3
215 nm
250 nm
290 nm
340 nm
100x Oil
1.3
215 nm
250 nm
290 nm
340 nm

 

Transmitted light microscopy: The Axiovert 200M has DIC and Phase Contrast on all objectives.  The high resolution color camera can be used for imaging fixed samples (e.g. histological preparations) and for time-lapse recordings of living cells. A stage heater is available for imaging living cells at a set temperature.

Fluorescence microscopy: The filter sets allow imaging of a wide variety of fluorescent indicators such as Alexa-Fluors, Calcium-Green, Cy2, Cy3, Cy5, DAPI, Fluorescein (FITC), FM1-43, Fluo-3, Fluo-4, GFP, CFP, YFP, Hoechst 33258, Rhodamine, Texas Red, TRITC, and others.  The stage heater and the sensitive monochrome camera make this microscope particularly well suited for imaging dynamic patterns of fluorescence in living cells.

Filter sets on the Axiovert 200M:

1) DIC:  Analyzer for DIC
2) DAPI: Chroma set 31000V2, Excitation 325-375 nm, Emission 435-485 nm (bandpass filter).
3) FITC: Chroma set 41001, Excitation 460-500 nm, Emission 510-560 nm (bandpass filter).
4) RHOD: Chroma set 41002c, Excitation 530-560 nm, Emission 590-650 nm (bandpass filter).
5) Cy5: Chroma set 41024, Excitation 590-650 nm, Emission 665 nm and above (long pass filter).

Also available upon request:

6) GFP: Chroma set 41017, Excitation 450-490 nm, Emission 500-550 nm (bandpass filter).
7) CFP: Chroma set 31044v2, Excitation 426-446 nm, Emission 460-500 nm (bandpass filter).
8) YFP: Chroma set 41028, Excitation 490-510 nm, Emission 520-550 nm (bandpass filter).
9) CFP/YFP FRET: Chroma set 31052, Excitation 426-446 nm, Emission 520-550 nm.

 

The Zeiss LSM 710 Confocal Laser Scanning Microscope

Laboratories for Molecular Medicine, room 111

The Zeiss LSM 710 confocal laser scanning microscope is designed for imaging fluorescence, either in fixed or live samples.  A pinhole removes the out-of-focus light and allows investigators to acquire thin optical sections at various focal planes.  Stacks of images can be acquired for 3D visualization. 

The confocal module is mounted on a Zeiss Axio Observer Z1 inverted microscope with high quality objectives (listed below). A heated stage is available for imaging live samples at 37°C.  The microscope has multiple lasers for excitation at 405, 458, 488, 514, 561, 594, and 633 nm, and is equipped with a 34-channel QUASAR detector, which can be set to collect emitted light of any wavelength within the visible spectrum.   The QUASAR detector is capable of acquiring spectral information and its flexibility allows imaging of a broad range of fluorescent indicators, including the Alexa-Fluors, Calcium-Green, Cy2, Cy3, Cy5, DAPI, Fluorescein (FITC), FM1-43, Fluo-3, Fluo-4, GFP, GFP variants (BFP, CFP, EGFP, YFP), Hoechst 33258, Rhodamine, Texas Red, TRITC, and others.  Images are acquired with ZEN 2008 software and can be stored on a CD, DVD, or a flash drive via a USB connection.  The LSM images can be opened using a freely downloadable ZEN LE viewer (link).

Objectives on the 710:

Table of Objectives
Mag
Correction
NA
DIC
WD (mm)
Coverslip #
10x
Plan-Apochromat
0.45
--
2.0
0.17mm #1 1/2
20x
Plan-Apochromat
0.8
DIC
0.55
0.17mm #1 1/2
40x W
C-Apochromat
1.2
DIC
0.28
0.17mm #1 1/2
63x Oil
Plan-Apochromat
1.40
DIC
0.11
0.17mm #1 1/2

Theoretical XY Objective Resolution*: based on: Dxy=0.61*wavelength/NA

Mag
NA
~460 nm (Dapi)
~535 nm (Fitc)
~620 nm (Rho)
~720 nm (Cy5)
10x
0.45
625 nm
725 nm
840 nm
975 nm
20x
0.8
350 nm
410 nm
475 nm
550 nm
40x W
1.2
235 nm
270 nm
315 nm
365 nm
63x Oil
1.40
200 nm
230 nm
270 nm
315 nm

Theoretical Z Objective Resolution*: based on: Dz=wavelength/(NA*NA)

Mag
NA
~460 nm (Dapi)
~535 nm (Fitc)
~620 nm (Rho)
~720 nm (Cy5)
10x
0.45
2270 nm
2640 nm
3060 nm
3555 nm
20x
0.8
720 nm
835 nm
970 nm
1125 nm
40x W
1.2
320 nm
370 nm
430 nm
500 nm
63x Oil
1.40
235 nm
270 nm
315 nm
370 nm

 

Image Analysis

Laboratories for Molecular Medicine, room 111

MetaMorph software (version 7.0) is available for image analysis. This versatile software package allows for deconvolution (digital removal of out-of-focus light), 3-D reconstruction, brightness measurements, cell counting, colocalization analysis, fluorescence / brightfield overlay, FRET analysis, morphometry, motion analysis, particle tracking, and timelapse measurements.