Microscopes in Sidney Frank Hall

Sidney Frank Hall for Life Sciences

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








 




 

 








The Olympus FV-1000-MPE Multiphoton Microscope

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.

As of August 2017 we have added a special Second Harmonics Filter cube that allows direct visualization of unstained Collagen fibers either isolated (in tendons or digested)  or w/in tissue (cardiac for example).  The filter is a 405/40 allowing selection of a stimulation wavelength between ~780 and ~840 nm. 

 

 


 


Olympus FV3000 Confocal MicroscopeOlympus FV3000 Confocal Microscope

 

 

 

 

 

 

 

 

 

 

Olympus FV3000 Confocal Microscope

Sidney Frank Hall, room 111

This confocal microscope can acquire thin optical slices of fluorescent samples. It has an inverted microscope configuration and is equipped with a resonant scanner for fast optical sectioning and 4 detectors, two standard Metal Alkalide and two high sensitivity GaAsP detetors.  

  • LASER DIODE 405NM,CW 50MW
  • LASER DIODE 488NM,CW 20MW
  • LASER DIODE 561NM,CW 20MW
  • LASER DIODE 640NM,CW 40MW

 Objectives on the FV3000RS:

Mag
Correction
NA
HFW @ 1x
WD (mm)
Immersion Medium
UPlan Apochromat
0.04
10.18 mm
5.0
Air/Dry
UPlan Super Apochromat
0.4
1.27 mm
3.1
Air/Dry
UPlan Super Apochromat
0.75
0.600 mm
0.60
Air/Dry
UPlan Super Apochromat
1.05
0.424 mm
0.80
Silicone Oil
UPlan Super Apochromat
1.3
0.212 mm
0.30
Silicone Oil

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The Zeiss LSM 710 Confocal Laser Scanning Microscope

Sidney Frank Hall, room 113

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 viewer.

Objectives on the 710:

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

 


 

 

 

Zeiss LSM800Zeiss LSM800

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The Zeiss LSM 800 Confocal Laser Scanning Microscope
Sidney Frank Hall, Room 115

The Zeiss LSM800 is an upright Confocal Laser Scanning Microscope, which is well-suited for imaging microscope slides. This microscope can image fluorescence in thin optical slices and stacks of images can be acquired to examine fluorescent structures in 3D.  The system is based on a Axio Imager Z2 microscope and is equipped with a URGB laser module (Diode lasers: 405, 488, 561 and 640 nm), high-quality objectives (10x, 20x, 40x water, 40x oil, 63x oil) and three sensitive GaAsP detectors. 

Objectives on the LSM 800:

Mag
Correction
NA
DIC
WD (mm)
HFW @ 1x
10x
PlanNEOFL
0.3
none
5.2
(non-confocal obj)
20x
Plan Apochromat
0.8
DIC II
0.55
319.5 µm
40x Water
C Apochromat
1.2
DIC III
0.28
159.7 µm
40x Oil
Plan Apochromat
1.3
DIC III
0.21
159.7 µm
63x Oil
Plan Apochromat
1.4
DIC III
0.19
101.4 µm














The Zeiss Lumar V12 Fluorescence Stereomicroscope
Sidney Frank Hall, Room 113

The Zeiss Lumar V12 Fluorescence Stereomicroscope is capable of imaging transmitted light, reflected light, and fluorescence of larger samples (field of view is 1.4 x 1.1 cm). 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.

Lumar Filters

DAPI (485049):  excitation 365 nm, emission 445/50 nm* 

GFP (486038): excitation 470/40 nm, emission 525/50 nm

Rhod (486043): excitation 550/25 nm, emission 605/70 nm

 

* 000/00: the first number is the midpoint of the band pass filter, the second number is the bandwidth of the filter.  E.g. Rhod 605/70 collects emitted light from 570 - 640 nm.

 














The Zeiss Axiovert 200M Fluorescence Microscope
BioMed Center, room BMC 005

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 4.8.2 software. This microscope is well equipped for transmitted light microscopy and fluorescence microscopy.

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 Water
C Apochromat
1.2
none
DIC III
0.28
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 Water
1.2
235 nm
270 nm
315 nm
365 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 49028, Excitation 384-407 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 Luminescence Microscope
Sidney Frank Hall, Room 117

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.

 














The Philips 410 Transmission Electron Microscope
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.

 


 

Apreo VS SEM for serial block-face imagingApreo VS SEM for serial block-face imaging

 

 

 

 

 

 

 

 

Thermo Apreo VS SEM for serial block-face imaging

Sidney Frank Hall, room 109

* Please acknowledge NIH support.  E.g. in publications that made use of the SEM:  The Thermo Apreo VS SEM was purchased with a high-end instrumentation grant from the Office of the Director at the National Institutes of Health (S10OD023461). 

The Apreo Volume Scope (VS) is equipped for standard scanning electron microscopy, as well as serial-block face imaging.  For serial block face imaging, ultrathin sections are cut inside the microscope and series of images are acquired from the freshly cut block face, while the sections are discarded.  The automated system is capable of imaging large z-stacks at isotropic 10x10x10 nm resolution.  The series of images can be processed for 3D reconstruction of cells and tissues, with sufficient resolution to identify small vesicles, organelles and neural connections. 

Samples for serial block-face imaging need to be processed using an enhanced contrast staining method.  Please contact Paula Weston in the Molecular Pathology Core for assistance.  Protocols are available on the Molecular Pathology Core website (Link). 

Amira software is available for stack alignment, 3D reconstruction and 3D segmentation (free of charge).  The full Amira for EM package is available on a high-end workstation in Sidney Frank Hall, room 106.  This version includes modules for analyses of large EM stacks. 

 Amira Online.  You can also install and run Amira basic on your own computer (2 floating licenses):

- Install Amira 2019.1: https://www.fei-software-center.com/amira/packagedownload-af5d824bb/ 
- OR Install Amira 2019.2: https://www.fei-software-center.com/amira/amira-packagedownload-a38c3c38d/
- After installation, run the Amira program (e.g. click on desktop icon) 
- On first startup:  Activate Amira, FNP:   lm_tfs.brown.edu    Activate

To use Amira Online, you need to sign up on our Amira calendar.  For access to this calendar, please email [email protected] or [email protected].

 




Microtomes, Critical Point Dryer, and Sputter Coater
Sidney Frank Hall, Rooms 105, 106, 109

The facility maintains equipment for sample preparation, including a Reichert Ultracut E microtome for ultrathin sectioning, a Ladd Research Industries critical point dryer, an Emitech K100X glow discharge unit, and an Emitech K550 sputter coater. The microtome is 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.

 




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 the 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: [email protected] 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/.





















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.

 

 

Olympus FV3000 Confocal Microscope (will be installed early Nov. 2017).  This confocal microscope can acquire thin optical slices of fluorescent samples. It is equipped with a resonant scanner for fast optical sectioning.  The system will be installed in Sidney Frank Hall, room 111.