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Sidney Frank Hall for Life Sciences

Room 106, 107, 109, 111, 113, 115, 117
185 Meeting Street
Providence, Rhode Island 02912

          Sidney Frank Hall

- Multiphoton Microscope
- Transmission Electron Microscope
- Scanning Electron Microscope
- Microtomes, Critical Point Dryer, and Sputter Coater
- Fluorescence Microscope
- Fluorescence Stereomicroscope
- Luminescence Microscope
- Confocal Laser Scanning Microscope (Leica)
- Confocal Laser Scanning Microscope (Zeiss)
- Image Analysis

The Olympus FV-1000-MPE Multiphoton Microscope

TEM

Sidney Frank Hall, room 106b

In April 2013, the Leduc Bioimaging Facility set up an Olympus FV1000-MPE multiphoton laser scanning microscope (also referred to as a ‘two-photon’ or ‘2P’ microscope).  This microscope is designed for high-resolution imaging of fluorescence at depths that are inaccessible to standard fluorescence or confocal microscopes.  The multiphoton microscope excites fluorescent indicators solely in the plane of focus and stacks of images can be acquired for 3D reconstruction and visualization.  The multiphoton microscope is based on an Olympus BX61wi upright microscope and is equipped with a Mai Tai HP tunable laser (690-1020 nm).  A SIM scan unit is available for simultaneous photostimulation with a 405 nm laser.  In addition, 458, 488, 515, 559, and 635 nm laser lines are available for sequential photoactivation and imaging.  The microscope is equipped with four non-descanned detectors (2 PMTs and 2 GaAsP detectors), an encoded Prior Z deck with a scanning stage, and 10x (NA 0.30, WD 3.3 mm), 25x (NA 1.05, WD 2 mm), and 60x (NA 1.00, WD 2 mm) objectives.  An analog input box with mapping and multipoint software is available for combined imaging and electrophysiology experiments.

 

The Philips 410 Transmission Electron Microscope

TEM

Sidney Frank Hall, room 105

The Philips 410 transmission electron microscope is equipped with a1k x 1k Advantage HR CCD camera from Advanced Microscopy Techniques (AMT). Images are acquired and analyzed with AMT's imaging software.

 

The Hitachi 2700 Scanning Electron Microscope

SEM

Sidney Frank Hall, room 109

The Hitachi 2700 Scanning Electron Microscope is equipped with a lanthanum hexaboride gun. Images up to are collected and analyzed with a Quartz PCI digital imaging system.

 

Microtomes, Critical Point Dryer, and Sputter Coater

Sidney Frank Hall, room 105, 106, 109

The facility maintains equipment for sample preparation, including a Reichert Ultracut E microtome and a Sorvall MT 5000 microtome for ultrathin sectioning, a Ladd Research Industries critical point dryer, and an Emitech K550 sputter coater.  The microtomes are used for cutting 1-5 um sections for light microscopy and 60-90 nm sections for transmission electron microscopy.  The critical point dryer and sputter coater are used to prepare samples for scanning electron microscopy.

 

The Zeiss Lumar V12 Fluorescence Stereomicroscope

Lumar

Sidney Frank Hall, room 113

The Zeiss Lumar V12 Fluorescence Stereomicroscope is capable of imaging transmitted light, reflected light, and fluorescence of larger samples (0.1-10 mm range). The microscope has a motorized focus and zoom, with foot pedals for hands-free operation.  The motorized filter turret allows one to collect brightfield and multiple colors of fluorescence in timelapse mode.  The microscope is equipped with filters for imaging DAPI, FITC / GFP, and RHOD and has a high NA objective (ApoLumar S 1.2x) and sensitive monochrome camera (AxioCam MRm), which makes this system suitable for imaging low levels of fluorescence. 

The Zeiss Axiovert 200M Fluorescence Microscope

200M

Sidney Frank Hall, room 113

 The Zeiss Axiovert 200M Fluorescence Microscope is a motorized inverted microscope with an AxioCam MRc5 color camera and a Hamamatsu ORCA-ER monochrome camera controlled by AxioVision 4.4 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 (Plan Neo 5x, 10x, 20x, 40x dry, 40x oil, and 100x oil).  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 Leica TCS SP2 AOBS Confocal Laser Scanning Microscope

Leica SP2

Sidney Frank Hall, room 115

The Leduc Bioimaging Facility houses a Leica TCS SP2 AOBS spectral confocal microscope. This filter-free confocal microscope is capable of imaging the spectum of a fluorescent indicator. It is equipped with nine laser lines throughout the visible spectrum: 405nm, 458nm, 476nm, 488nm, 496nm, 514nm, 543nm, 594nm, and 633nm. Four detection channels are available, allowing simultaneous imaging of multiple fluorescent indicators. In each of the four channels, fluorescence can be imaged between two adjustable wavelengths. A fifth channel is used to collect brightfield or DIC images. Leica's confocal software, LCS version 2.5, is used for image acquisition and analysis. This software package includes numerous analysis options, including intensity measurements, colocalization analysis, and 3D reconstruction.

Objectives on the SP2:

Table of Objectives
Mag
Correction
NA
DIC
WD (mm)
Coverslip #
10x
HC PL APO
0.4
-
2.2
0.17mm #1 1/2
20x
HC PL APO
0.7
C
0.59
0.17mm #1 1/2
63x Water
HCX PL APO
1.2
D
0.22
0.17mm #1 1/2
63x Oil
HC PL APO
1.40-0.6
E
0.10
0.17mm #1 1/2
100x Oil
HC PL APO
1.40-0.7
D
0.09
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
0.4
700 nm
815 nm
945 nm
1100 nm
20x
0.7
400 nm
465 nm
540 nm
630 nm
63x Water
1.2
235 nm
270 nm
315 nm
365 nm
63x Oil
1.4
200 nm
230 nm
270 nm
315 nm
100x Oil
1.4
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
0.4
2875 nm
3345 nm
3875 nm
4500 nm
20x
0.7
940 nm
1090 nm
1265 nm
1470 nm
63x Water
1.2
320 nm
370 nm
430 nm
500 nm
63x Oil
1.4
235 nm
270 nm
315 nm
370 nm
100x Oil
1.4
235 nm
270 nm
315 nm
370 nm

*Resolution numbers are approximate, assuming ideal sample and fluorochrome conditions, proper coverslip, and pinhole settings. Values are listed for general information purposes.

 

The Zeiss LSM 510 Meta Confocal Laser Scanning Microscope

Zeiss LSM 510meta

Sidney Frank Hall, room 111

The Zeiss LSM510 Meta Confocal Laser Scanning Microscope is built on an Axiovert 200M inverted microscope.  This microscope can image fluorescence in thin optical slices and stacks of images can be acquired to examine fluorescent structures in 3D.  The microscope is equipped with seven laser lines throughout the visible spectrum: 405 nm, 458 nm, 477 nm, 488 nm, 514 nm, 561 nm, and 633 nm.  It has one detector for brightfield imaging, two PMT detectors for fluorescence imaging, and one Meta detector for spectral fluorescence imaging.  The Meta detector allows one to quickly collect spectral information that can be used for subtracting background fluorescence or for linear unmixing of overlapping dyes.  The microscope is well-equipped for various in vivo techniques including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and fluorescence resonance energy transfer (FRET).

Objectives on the 510 meta:

Table of Objectives
Mag
Correction
NA
DIC
WD (mm)
Coverslip #
5x
Plan Apochromat
0.16
-
12.1
0.17mm #1 1/2
10x
Plan Apochromat
0.45
DIC II
2.0
0.17mm #1 1/2
20x
Plan Apochromat
0.8
DIC II
0.55
0.17mm #1 1/2
40x Water
C Apochromat
1.2
DIC III
0.28
0.17mm #1 1/2
63x Oil
Plan Apochromat
1.4
DIC III
0.19
0.17mm #1 1/2
100x Oil
alpha Plan-Fluar
1.45
DIC III
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
0.16
1755 nm
2040 nm
2520 nm
2930 nm
10x
0.45
625 nm
725 nm
840 nm
975 nm
20x
0.8
350 nm
410 nm
470 nm
550 nm
40x Water
1.2
235 nm
270 nm
315 nm
365 nm
63x Oil
1.4
200 nm
230 nm
270 nm
315 nm
100x Oil
1.45
195 nm
225 nm
260 nm
300 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
0.16
17970 nm
20900 nm
24220 nm
28125 nm
10x
0.45
2270 nm
2640 nm
3060 nm
3555 nm
20x
0.8
720 nm
835 nm
970 nm
1125 nm
40x Water
1.2
320 nm
370 nm
430 nm
500 nm
63x Oil
1.4
235 nm
270 nm
315 nm
370 nm
100x Oil
1.45
220 nm
255 nm
295 nm
340 nm

*Resolution numbers are approximate, assuming ideal sample and fluorochrome conditions, proper coverslip, and pinhole settings. Values are listed for general information purposes.

 

The Luminescence Microscope

Sidney Frank Hall, room 107,

Luminescence microscope photon imaging example

The Leduc Bioimaging Facility houses a Photon Imaging Microscope capable of imaging bioluminescent and chemiluminescent indicators on a cellular level. In contrast to fluorescent indicators, luminescent indicators do not require an exciting light beam, thus circumventing problems of photobleaching and phototoxicity. Luminescent indicators such as luciferin (detects luciferase expression), luminol (detects reactive oxygen species), or aequorin (detects intracellular calcium) can be imaged in living cells for days on end, without disturbing the cells under investigation. The Photon Imaging Microscope consists of a Zeiss Axiovert 100TV inverted microscope and a Photometrics Cascade II EMCCD camera.

 

Image Analysis

Sidney Frank Hall, room 106c

FluidVis.  Collaborations between Brown’s Computer Science Department, the Center for Computation and Visualization (CCV) and the basic science departments in Division of Biology and Medicine have led to the development of the research software CaveVOX, a fully-immersive interactive three-dimensional visualization application that runs in the Brown Cave at 180 George Street. CaveVOX has been in use for several years and helped understand some 3D stacks more quickly and led to discoveries overlooked with desktop visualization tools. FluidVis is a new software that provides a semi-immersive experience similar to CaveVOX and is available for use now in the Leduc Bioimaging Facility.  FluidVis runs on hardware acquired through an OVPR seed grant awarded to Professor Kristi Wharton.  Both CaveVOX and FluidVis create an intuitive virtual environment for analyzing stacks of images in 3D and are ideally suited for the analysis of samples with considerable 3D complexity.  For example, networks of neural connections can be difficult to analyze in two dimensions, but stand out bright and clear in a 3D environment.  A natural user interface makes changing viewpoints and exploring or tuning visualization settings easy and fast.

For a demo or training on FluidVis, please contact Robbert Creton by phone at 401-863-9646 or by email at:  Robbert_Creton@brown.edu.   Please feel free to bring a stack of images for the training.  The images should be in TIF format, one series per folder, with sequentially numbered names.  For additional information about CaveVOX and 3D visualization systems at the Center for Computation and Visualization, please visit the CCV website at:  http://www.ccv.brown.edu/ .  For more information about FluidVis, please visit http://www.fluiditysoftware.com/.

Brain in the CaveVesicle in Cave

Investigator uses CaveVOX to explore 3D datasets acquired by confocal microscopy

Sidney Frank Hall room 106B also houses two workstations for image analysis:

1) Metamorph. The first workstation contains MetaMorph software (version 7.0), which can be used 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.

2) Confocal Software. The second workstation contains image analysis software for the Zeiss Confocal Microscope (LSM Imager) and Leica Confocal Microscope (LCS) that can be used for image viewing, export of TIF images, fluorescence / brightfield overlays, quantitative measurements, colocalization analysis, and 3D reconstruction.