Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

The overall goal of this procedure is to perform single plane illumination microscopy using a light sheet excitation module for inverted microscopes and apply it to three dimensional cell spheroids. This is accomplished by first using a pipette to transfer a cell spheroid from the well of a microtiter plate to a second well to incubate following washing. The S spheroid is then introduced to a rectangular capillary.

The second step is to mount the light sheet illumination module to the base plate of the inverted microscope positioning table. Next, the capillary containing the steroid is placed in a special sample holder for microscopy. The final step is to set the matching between the light sheet and the focal plane of the microscope objective lens in the detection path.

Ultimately, three dimensional fluorescence measurements can be performed by variation of the focal plane within the cell spheroid to gain information of single images or Zacks concerning the uptake of drugs or the structure of the S spheroid. The main advantage of this technique over existing light sheet microscopes is that it is cost efficient and highly versatile as it consists of a module that can be adapted to an existing microscope and provides a large degree of freedom for research. The implications of this technique extend towards research of drugs and agents used for therapy and diagnosis, in particular, since adaption of the microfluidic system permits investigation of rapid processes under flow conditions.

Now this excitation method in combination with any detection technique can provide insight into live cell steroids at low light doses. It can also be applied to other living systems such as cipro fish, artila embryos, as well as optical CLE tissue samples. To begin, add 50 microliters of heated 1.5 aeros in culture medium into each well of a 96 well plate and let it solidify for one to two hours.

Then seed about 150 MCF seven breast cancer cells in each well of the prepared 96 well plate gross S for five to seven days, up to a diameter of about 200 to 300 micrometers in an incubator at 5%carbon dioxide and 37 degrees Celsius. Next, incubate the cell steroids with the anthracycline antibiotic doxorubicin hydrochloride for six hours at a concentration ranging between two micromolar and eight micromolar in culture. Medium Following incubation, wash the cells OID with culture, medium or Earl's balance salt solution prior to microscopy.

Use an inverted microscope and mount the single plane illumination microscopy or SPM excitation module to the base plate of the positioning table. Equip the microscope with a 10 times or 20 times magnifying microscope objective lens and an appropriate long pass filter in the detection path of the microscope. Then mount an integrating camera to the detection port of the microscope.

Use a parallel collated beam of a laser or a laser diode with an excitation wavelength of preferentially 470 nanometers and apply it to the spin module. Next, rotate the cylindrical lens by 90 degrees, which transfers the light sheet into a vertical position for an axial adjustment of the beam waist of the light sheet. Place a capillary containing a liquid with a fluorescent dye in the sample holder.

Attach the sample holder with the capillary to the positioning table of the microscope. Align the position of the capillary such that it is centered and the beam waist of the light sheet is in focus. Adjust the beam waist by variation of the axial position of the cylindrical lens itself.

Turn back the lens after adjustment. Place the cell steroid separately or in groups within rectangular bo silicate glass capillaries with an inner cross section of 600 by 600 micrometers and a wall thickness of 120 micrometers. To achieve this, take the empty capillary upright with thumb and middle finger and seal the upper opening with forefinger.

Bring the lower opening close to the cell spheroid in its surrounding liquid release forefinger from the upper opening liquid with the steroid in. It will be soaked in immediately by capillary forces. Adjust the position of the S spheroid within the filled capillary by gravitation.

Alternatively, apply the steroid to the capillary via pipetting. Take care that the opening of the tip of the pipette is on the one hand big enough for the S spheroid size and on the other hand, less or equal in size to the inner diameter of the capillary. Next place the capillary containing the S spheroid in a special sample holder from microscopy.

Attach the sample holder with the capillary to the positioning table of the microscope and align the position of the cell steroid such that it is centered and focused. Set the matching between the light sheet and the focal plane of the microscope objective lens in the detection path by adjusting the position of the reflection mirror and the cylindrical lens. Fill the capillary with fetal calf serum or FCS for 30 minutes to support later cellular adhesion of a cell steroid to the inner glass surface.

Let the remaining FCS coating in the depleted capillary dry out for at least 12 hours after introducing the cell steroid to the FCS coated capillary. As before, leave the capillary in the incubator for an additional two to four hours to cause cellular adhesion of the steroid. Next, set up the flx part of the microfluidic system including a bubble trap.

Use a peristaltic pump to fill the flx of the tubing with culture medium containing the fluorescent dye drug or agent. First clamp the bubble free capillary to the efflux tubing and then clamp the other side of the capillary to the drain tubing. Tune the liquid temperature of the water bath to the desired value.

Then adjust the peristaltic pump to the desired pump velocity. Collect the pumped liquid into a recipient with an open loop setup for data acquisition and analysis. Set the laser power and the integration time for the image acquisition.

Set the increment for the ZS stack to a value between five and 10 micrometers, which is preferable for three dimensional data analysis. As the light sheet thickness is about 10 micrometers proceed to perform measurements of single images or Zacks by variation of the focal plane within the cell steroid. A Zack scan of an mc seven cell steroid previously incubated with eight micromolar doxorubicin for six hours is shown.

The images give detailed information about the cellular uptake and distribution of doxorubicin and its degradation. Product within the outer cell layer of the steroid red fluorescent doxorubicin is mainly localized in the nucleus, whereas in inner sphe roid areas, green fluorescent submitted by a degradation product becomes dominant in the cellular membrane. The uptake of the chemotherapeutic drug doxorubicin in native mc seven human breast cancer cell S is depicted for a single cell layer selected by spm.

Upon application of two micromolar and culture medium within the flow system, red and green fluorescence of doxorubicin and its degradation product increased continuously upon application of two micromolar doxorubicin in culture medium within the flow system, red and green fluorescence of doxorubicin and its degradation product increased continuously. Once mastered, it takes only a few minutes from mounting the light sheet elimination module until recording fluorescent images of 3D samples. When measuring and the flow condition, it's most important to assure that the sample adhere to the inner surface of the capillary and to avoid air bubbles reaching the sample which might detach it from the capillary.

After watching this video, you should have a good understanding of light sheet illumination microscopy with the versatile module adaptable to various microscopes.