Con­focal laser scan­ning mi­cro­scope

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The con­focal laser scan­ning mi­cro­scope uses a trick with light. With nor­mal light mi­cro­scopes as well as with the fluor­es­cence mi­cro­scope, the en­tire spe­ci­men is il­lu­min­ated. However, things are dif­fer­ent with a con­focal mi­cro­scope. Here, a laser is used to scan the area of in­terest point by point. Soft­ware then com­piles the in­form­a­tion into an im­age. The fluor­es­cent light is fo­cused by a pin­hole dia­phragm, which fades out the areas out­side the fo­cus. The end res­ult is a sharper im­age without dis­tract­ing over­lays.

Other meth­ods can also be ap­plied so that more de­tail can be seen on the im­ages at the same res­ol­u­tion. For this pur­pose, the con­focal mi­cro­scope has two ex­ten­sions: an Airy­scan de­tector and the ELYRA high-res­ol­u­tion sys­tem with the SR-SIM and PALM or dSTORM tech­niques.

Airyscan

One prob­lem with a con­focal mi­cro­scope is that there is too little light. An al­most closed pin­hole al­lows for a strong fo­cus. However, it is also nearly dark be­cause hardly any light gets through. The strength of the con­focal mi­cro­scope thus be­comes a weak­ness the more the pin­hole is closed. Airy­scan solves this prob­lem. It con­sists of sev­eral de­tect­ors ar­ranged in a circle and re­sembles a com­pound eye. Each in­di­vidual de­tector has the dia­meter of an al­most closed pin­hole and thus “sees” its sec­tion as sharply as pos­sible. Be­cause there are 32 such de­tect­ors in our ver­sion of the Airy­scan, each with a spe­cific fo­cal point, there is enough light in total to be able to de­tect something.

Airy­scan can achieve 1.7-fold the res­ol­u­tion of stand­ard mi­cro­scopy and in­creases the res­ol­u­tion limit to 140 nm.

ELYRA

The ELYRA sys­tem in­cludes two tech­niques for high-res­ol­u­tion im­ages. The first is called Su­per­res­ol­u­tion Struc­tured Il­lu­min­a­tion (SR-SIM). It is based on the Moiré ef­fect. This is the ef­fect cre­ated when two striped pat­terns over­lap. For ex­ample, when someone wears a striped shirt on TV, the audi­ence in front of the screens sees a flicker of the pat­tern. The flicker is caused by the crossed over­lap­ping of the shirt stripes with the im­age lines of the cam­era. This ef­fect can be cal­cu­lated.

The SR-SIM method works by il­lu­min­at­ing the bac­teria with a pat­tern of stripes and then tak­ing a pic­ture. The Moirè ef­fect then oc­curs where there are nat­ural “stripe” struc­tures in the pre­par­a­tion. This is re­peated sev­eral times with dif­fer­ent po­s­i­tions. Soft­ware can then cal­cu­late back to the nat­ural struc­tures of the spe­ci­men from the Moirè pat­terns present in the im­ages and thus cre­ate a co­hes­ive im­age. In the best case, it is pos­sible to re­cog­nize struc­tures with a res­ol­u­tion of up to 100 nm (i.e. it makes it pos­sible to see twice as well as with a con­ven­tional light mi­cro­scope).

The second tech­nique of the ELYRA ex­ten­sion on the con­focal mi­cro­scope of our In­sti­tute is photo-ac­tiv­ated loc­al­iz­a­tion mi­cro­scopy (PALM). This is a la­bor­i­ous method be­cause the sample must be spe­cially pre­pared for this. It is based on fluor­es­cence and takes ad­vant­age of the fact that if not all fluor­es­cence mo­lecules light up at the same time, the centre of each light spot cor­res­ponds to the po­s­i­tion of a single fluor­es­cent mo­lecule. The sample is pre­pared in such a way that, if pos­sible, no two ad­ja­cent mo­lecules light up at the same time. These light points are then meas­ured and dis­played in a table. The out­put of this method is there­fore not an im­age but rather a table with the co­ordin­ates of the light points. However, a pic­ture can be cal­cu­lated from this if ne­ces­sary. The­or­et­ic­ally, a res­ol­u­tion of up to 20 nm is pos­sible with PALM.