How to buid a super-resolution microscope

A super-resolution microscope, PALM (sometimes called STORM), provides a fluorescence image with high spatial resolution on the order of 10 nm. The set-up of PALM is very simple; therefore, you can easily build it by yourself. A home-made PALM system costs only 20-40 % of a commercial system. Here we describe how to build your own super-resolution microscope.
The right figure shows the block diagram of the optical system of PALM. Co-linearly overlapped laser beams are introduced to the excitation illumination port of a microscope and the fluorescence signal from the sample is imaged onto a CCD camera through optical filter. Details on the components of the system and the procedure to build it are below.

Equipment

Microscope PALM is built based on an inverted microscope. Any commercial microscope can be used. If you don't have to take phase-contrast or DIC images simultaneously with the super-resolution image, the related equipments such as a transmission illumination is not necessary. PALM is sensitive to the drift motion of the sample; therefore, it is recommended to use a sample stage with high mechanical stability. We use a home-made stage with a XY translation stage having a large aperture (TSD-1202CH from Sigma Koki, or similar).
Objective lens
The selection of the objective is important. The pixel size should be correspond to ca. 100 nm at the sample plane. For example, the actual pixel size of the camera is 16 um, the magnification should be 160. If you do not have an objective with an appropriate magnification factor, a relay lens pair should be placed in front of the camera to adjust the magnification. A numerical aperture should be as high as possible for the highest resolution and collection efficiency. We use a 100x objective with 1.4NA and a magnification lens installed in a microscope body.
Camera
Highly sensitive camera is necessary for high spatial resolution and acquisition speed. An EMCCD camera is recommended, because it can detect the weak fluorescence from a single dye molecule at a high signal-to-noise ratio. We use iXon+ from Andor and Cascade II from Roper.
Light source
Laser systems for activation, fluorescence excitation, and deactivation are necessary. The wavelength should be selected considering the absorption band of the dye used in the experiment. The power density for the excitation is more than several kW/cm2 at the sample, which corresponds to the laser power of 10 mW for an area of 10 x 10 um. The power for the activation and deactivation is greatly dependent on the conversion efficiency of dye. A dichroic mirror and a band-pass filter should be selected considering the wavelengths of the light sources and the fluorescence of the dye. The filters are available from Semrock, Chroma Technology, and so on.
Shutter
The timing of the illumination is controlled by shutter, AO, or EO devices. The timing system should be driven by a pulse generator (from Quantum Composers, Stanford Research Systems, or similar).
Environment
The microscope should be mounted on a vibration-isolated optical bench to avoid the blurring of the fluorescence image due to the vibration. The whole system should be placed in a temperature-stabilized room.
Optics
Other optics such as mirrors and lenses are available from Sigma Koki, Thorlabs, Newport, and so on. We use aluminium and silver mirrors for UV and visible regions, respectively. The mirrors and lenses should be placed using kinematic mounts.

Setup of optics

 1. Mount laser heads tightly on a table. If AO or EO devices are used for the illumination control, set up them at this time. Adjust the beam diameter with an expander if it is necessary. The beam diameter determines the size of the field of view of the PALM measurement.
 2. Overlap the beams of the activation, excitation, and deactivation lasers co-linearly by dichroic mirrors, DM1 and DM2.
 3. Mount the microscope body onto the optical bench and install a camera. Place a filter set (DM3 and BP) and remove OBJ. (Optional: Insert a telescope consisting of L2 and L3 to adjust the magnification factor.)
 4. Input the combined beam into the excitation port of the microscope using steering mirrors M1 and M2. The distance between OBJ and M1 should be twice of the focal length of L1 (L1 is not placed at this stage). Align the beam to pass the centers of the excitation port and the objective mount port. The laser beam should be directed from the objective port to the ceiling at a right angle by adjusting M1 and M2.
 5. Set OBJ and a fluorescent thin film, which was prepared by spin casting a poly(vinyl alcohol) solution containing a fluorescent dye with the absorption band at the wavelength of the laser.
 6. Observe the sample by the camera set on the microscope. A bright spot should be observed at the center of the field of view. If it is not the case, adjust the position and the incidence angle of the laser beam.
 7. Place a lens, L1, in front of the excitation port of the microscope, which focuses the laser beam onto the back focal plane of OBJ. The collimated beam should be emitted from OBJ to the ceiling at a right angle. If it is not the case, adjust the position of L1.
 8. A fluorescence image of the sample with Koehler illumination mode is observed. If the illuminated area is not the center of the field of view, adjust the beam position by M1 and M2. If you want to adjust the shape of the illuminated area, put an aperture A between DM1 and M2. The total internal reflection (TIR) illumination is also possible by changing the angle of M1.

Setup of electronics

 1. Place shutters in the beam path to control the timing of the illumination.
 2. The camera exposure and the laser illumination should be synchronized by a trigger signal from a pulse generator. Wire the trigger inputs of the camera and lasers to the output of the pulse generator.
 3. Program the sequence of the trigger signal. An example of the pulse sequence is below, where the high state corresponds to he open state of the shutter. The pulse length should be changed dependent on the measurement condition. The excitation laser is operated in CW.


In this example, the camera and lasers are driven by the pulse generator. If the camera outputs timing signals, it can be used as the master clock for the illumination control.
 4. Check the output pulse by an oscilloscope and test the synchronization of the camera and lasers.

Image acquisition

 1. Under construction

Data analysis

 1. Under construction


If you have questions and comments, please send an e-mail to hiroyuki.aoki(_at_)j-parc.jp .