IGC

dSTORM | Reservations

  • dSTORM microscope

  • dSTORM (direct STochastic Optical Resconstruction Microscopy) is a super-resolution microsocpe custom-built by the Advanced Imaging facility.

    Location : Bartolomeu Dias wing, UIC Room 6

    Microscope: Nikon Ti

    Year Installed: 2012

    Camera: Hamamatsu Flash Orca 4 2048*2048*

    Optics: Fluorescence + STORM

    * - camera usualy works at 512*512 for higher frame rate and homogeneous field of view.


  • dSTORM (direct STochastic Optical Resconstruction Microscopy) is one of the super-resolution techniques based on Single Molecule Localization microscopy to go beyond the resolution limit of 200nm observed in regular widefields or confocal microscopes. This technique takes advantage of the blinking properties of some fluorophores, emiting light at different time intervals and consequently allowing distinction between fluorescent proteins within the same space. This technique, in optimal conditions, can achieve resolutions of 30 or 40 nm.

    dSTORM acquisition and reconstruction

    Van de Linde et al. 2011 Nature Protocols

    In dSTORM, blinking is induced with the use of high laser powers and a chemical buffer, that induces a triplet state in the fluorescent molecules. The triplet state is an intermediate "layer" where the electron is still at a high energy level but does not recover to the ground state, not emiting a photon. This process of going to the ground state with photon emission is stochastic, which may allows us to see different molecules even if they are in the same physical space below the resolution limit of normal microscopes.

Suggestion for description in "Materials and Methods":

Images were acquired on a custom made system based on a Nikon Ti microscope body, equiped with a Hamamatsu Flash ORCA 4.0, using the a Nikon 100x 1.45 NA Oil immersion objetive. A 561nm Coherent Genesis MX 500mW and a 642 nm Vortran Stradus 100mW lasers were used to excite Alexa 561 and Alexa 633, respectively. For maximum specificity, a Chroma BP 595/50nm filter was used for the acquisition of 561 excitation and a Chroma 640LP filter for 642 excitation were used. Images were acquired with MicroManager microscope control software.

Suggestion for "Acknowledgments":

We would like to thank the technical support of IGC's Advanced Imaging Facility (AIF-UIC), which is supported by the national Portuguese funding ref# PPBI-POCI-01-0145-FEDER-022122, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Fundação para a Ciência e a Tecnologia (FCT; Portugal).

Maintenance log:


Laser Unit

Laser
Excitation lines
Maximum power
Omicron LuxX
405 nm
60 mW
Coherent Genesis MX 488
488 nm
1000 mW
Coherent Genesis MX 561
568 nm
500 mW
Vortran Stradus
642 nm
110 mW

Available objectives

Below is a list of the objectives and filters available. On the dSTORM system, the filter cubes are set and changed through the computer as there are 2 sets of filters and need to be syncornized to avoid image problems.

Move mouse over me!
Magnification
Objective type
NA1
Immersion
Working distance (mm)
Resolution2 @525nm in XY (µm)
Image Pixel size3 (µm)
Link
10x
Plan Fluor
0.3
-
16
1.068
1.44
20x
Plan Apo λ
0.75
-
1
0.427
0.72
100x
Plan Apo λ
1.45
OIL
0.13
0.221
0.144

(1) NA - Numerical Aperture. For more information, follow this link.

(2) These values are the theoretical optical resolution limits depending on the NA of the objective and were calculated using the Resolution formula adapted to fluorescence as seen in the Olympus micro website: R = 0.61*λ/NA

(3) The pixel size saved automatically with the .ome.tif files. The values on this table were measured with a stage graticule, without the 1.5x auxiliary magnification. For more accurate measurements use these values instead.


Installed filtersets

Eyepiece filtersets

Fluorescence filterset
Excitation wavelength (λ)
Dichroich
Emission wavelength (λ)
DAPI
350/50
400
460/50
FITC
480/40
510
535/50
Cy3
545/25
565
605/70
Cy5
620/60
660
700/75

Camera filters

Fluorescence filterset
Emission wavelength (λ)
Description
name
DAPI
435/31
DAPI, Hoesche, etc.
1
GFP
528/48
GFP, FITC, Alexa Fluore 488, etc.
1
RFP
609/37
RFP, TRITC, mCherry, AF568, etc.
2
Dual 488/640
560/120-700/50
GFP or Alexa 488 and Cy5 or AF633
Dual 488/640
Quad
450/50-525/50-595/50-655lp
Quadruple filter
ZQuad

Spectra, lines & filters:


Setup

Turning system ON

  1. Turn on the lasers power bar.

    Lasers power bar

  2. Turn on the 488 or 561 or both lasers by pressing the on button on the back of the laser control box.

    Laser power control box

  3. Turn on the second power bar on the side of the table.

    Power bar number2

  4. Open the acquisition software, MicroManager and choose the latest stable configuration.

    MicroManager icon.


Turning OFF

  1. Close the acquisition software.
  2. Clean the objective and lower the Z position.
  3. Turn of the second power bar (see above for a picture).
  4. Turn of the lasers you used (488 or 561).
  5. Turn of laser power bar.
  6. Done!
  7. TAKE OUT EVERYTHING YOU BROUGHT!, eg, pipettes, slides, etc...

Extra Info

ImageJ/Fiji recognize and open DeltaVision .dv files, but you will need to use the Bio-Formats plugin. This plugin is available by default in the Fiji distribution.

To install the plugin in ImageJ, download the latest stable release of Bio-formats and drag it into the plugins folder. Once you start ImageJ again it will appear on the plugins menu. Go to "Plugins>Bio-formats>Bio-formats importer" or drag-and-drop a file in ImageJ/Fiji to open it.

Differential Interference Contrast (DIC)