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Laboratories for Molecular Medicine

Room 110, 110A, 111
70 Ship Street
Providence, Rhode Island 02903

 

 

 

 

 

- Fluorescence Microscope
- Confocal Laser Scanning Microscopes
- Image Analysis

 

The Zeiss Axiovert 200M Fluorescence Microscope

Laboratories for Molecular Medicine, room 110A

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

 

The Zeiss LSM 410 Confocal Laser Scanning Microscope

Laboratories for Molecular Medicine, room 111

The Zeiss LSM 410 confocal laser scanning microscope is equipped with three lasers: a UV laser for excitation at 364 nm, a krypton-argon laser for excitation at 488, 568, and 647 nm, and a helium-neon laser for excitation at 543 nm. The microscope has two separate detection channels with the ability to collect up to 3 channels sequentially. It also has a large number of filter sets that allow imaging of numerous fluorescent indicators,such as 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. Data acquisition is carried out using Phoenix software from Microcosm and images can be stored on CD or DVD media.

Objectives on the 410:

Table of Objectives

Mag	Correction	NA	DIC	WD (mm)	Coverslip #
5x	Plan Neofluar	0.15	-	13.6	0.17mm #1 1/2
10x	Plan Apochromat	0.45	DIC	2.0	0.17mm #1 1/2
25x W/O/G	LCI Plan Neofluar	0.8	DIC	0.21	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)
5x	0.15	1870 nm	2175 nm	2520 nm	2930 nm
10x	0.45	625 nm	725 nm	840 nm	975 nm
25x W/O/G	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)
5x	0.15	20445 nm	23780 nm	27555 nm	32000 nm
10x	0.45	2270 nm	2640 nm	3060 nm	3555 nm
25x W/O/G	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 109

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.