Robertson, John L.

Wilson, Charles L.

English

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 22 Article 13 1968 Microirradiation of Living Fungal Cells with a Laser John L. Robertson Charles L. Wilson Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Cell Biology Commons Recommended Citation Robertson, John L. and Wilson, Charles L. (1968) "Microirradiation of Living Fungal Cells with a Laser," Journal of the Arkansas Academy of Science: Vol. 22 , Article 13. Available at: https://scholarworks.uark.edu/jaas/vol22/iss1/13 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. 64¦iArkansas Academy of Science Proceedings, Vol. 22, 1968Microirradiation of LivingFungal Cells with a Laser 1John L. Robertson and Charles L. WilsonINTRODUCTIONThe introduction of the laser into biology has provided a toolwith which new questions can be asked, and possibly answered. Ithas proven to be a useful instrument for microsurgery because avery small area of the cell can be destroyed with little apparentdamage to the remaining portions. There is evidence that laserradiation can be used as a mutagen in much the same manner asx-ray or other types of radiation. This paper discusses the methodsinvolved in preparation of living fungal materials for irradiation witha laser beam and describes some responses of irradiated cells. Toour knowledge, experiments of this nature have not been performedpreviously with the fungal cell.MATERIALS AND METHODSMaterial to be irradiated was prepared by inoculating agar-dipped slides wth blocks of agar containing fungal mycelium. In-oculated slides were incubated at 24°C for 15 to 18 hours in amoist petri dish. Agar-dipped slides were prepared as follows: sterileslides, .96 to 1.06 mm in thickness, were dipped into hot Difco OakWilt agar in a sterile petri dish and allowed to become well covered.They were then held on end to drain off excess agar, leaving onlya thin film on the slide. After inoculation and incubation of thefungus the excess agar was scraped off the slide leaving a smallsquare of the agar with fungal hyphae. A cover slip, no. 0, was thenplaced on the slide and all edges of the coverslip were sealed withparaffin.A Leitz phase-contrast microscope with Heine condenser wasused in conjunction with a TRG bio-coupling model 153A lasersystem. Shots were fired through a Leitz 70x oil fluorite objective.The laser has a maximum power input of 230 joules and could befired once every minute with a pulse duration of 150 u sec. The laserwas aimed by use of a set of crosshairs in the ocular of the micro-scope. Because of the large size of its cells, Basidiobolus ranarunEidam was the fungus used in this study.RESULTSThe results of microirradiation are affected by the activityof the cell at the time of irradation. Ifthe cell is dividing, a muta-:Published with the approval of the Director of the Arkansas Agricultural Experiment Station.64Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 13Published by Arkansas Academy of Science, 196865|Laser Irradiation of Fungal Cellsgenie effect may be obtained. When a shot is directed into thechromatin at telophase, a portion of the chromatin is destroyed withthe resulting formation of a nucleus which is much reduced in size.If a shot is directed into the spindle during division, no cellwall subsequently forms, so that a cell with two nuclei is produced.When a nucleolus is irradiated at one end (Fig. 1) a darkenedarea appears at the focal point of the beam. After a period of about2 minutes the nucleolus can be seen to constrict at the point ofirradiation (Fig. 2). Approximately two hours after tihe initialshot, a portion of the nucleolus may separate from the main body(Fig. 3). The separated portion may fragment into small bodieswhich remain within the nuclear envelope. (Fig. 4).The elongation of a cell can be arrested by irradiation of thetip. This, however, must be done with care because it is quite possibleto burn a hole in the cell wall permitting the cytoplasm to escapethrough it. These holes generally range in size from 1 to 3 u (Fig. 5).DISCUSSIONTh<e amount of the cell damaged, and the type of damage doneby laser irradiation depends on several factors. The amount ofenergy input and the size of the aperture affects the size and in-tensity of the beam in the TRG system. Magnification also affectsthe size of the damaged area: the higher the magnification, thesmaller the damaged area. Ifthe microscope is out of focus, a muchlarger portion of the cell will be damaged. The depth at which thecell is embedded in the agar also determines how effective a shot willbe; this is one reason for the importance of the thin film of agaron the slide. Thin preparations are absolutely essential in order toobtain good results with laser irradiation through the microscope. Italso encourages the fungus to grow in one plane, thus allowing entirecells to be brought into focus at the same time.The reaction of the nucleolus when irradiated indicated thatthere is something which bounds its contents. Although no membranehas been found around the nucleolus with ultrastructure studies, theevidence presented here appears to indicate that one may be present.Such a membrane might not have the generally accepted unit mem-brane structure and therefore, would not be detectable by the methodsgenerally used in electron microscopy. A membrane that is highlyelastic and capable of repairing itself following breakage is suggestedby responses of the nucleolus to microirradiation.With laser radiation there is a possibility of increasing or re-ducing ploidy by selectively destroying the spindle or part of thenuclear material.65Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 13https://scholarworks.uark.edu/jaas/vol22/iss1/1366Arkansas Academy of Science ProceedingsLaser irradiation should provide a valuable tool for studies tobetter understand the fungal cell. The opportunity to selectivelydestroy parts of the cell should allow morpihpgenic and geneticstudies to be made that were hitherto impossible.DESCRIPTION OF FIGURESMicroirradiation of Basidiobolus Ranarum With LaserFig. 1. 650x Nucleolus shortly after irradiation. Arrow at pointof irradiation.Fig. 2. 650x. Constriction of same nucleolus (arrow) 2 minutesafter irradiation.Fig. 3 650x. The point of initial irradiation (R) at the far leftand constriction of nucleolus (c).Fig. 4. 1500x. Fragments (F) of nucleolus within the nuclearenvelope.Fig. 5. 1500x. Irradiated area at the tip.66Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 13Published by Arkansas Academy of Science, 1968

Microirradiation of Living Fungal Cells with a Laser

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 22 Article 131968Microirradiation of Living Fungal Cells with aLaserJohn L. RobertsonCharles L. WilsonFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Cell Biology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu, ccmiddle@uark.edu.Recommended CitationRobertson, John L. and Wilson, Charles L. (1968) "Microirradiation of Living Fungal Cells with a Laser," Journal of the ArkansasAcademy of Science: Vol. 22 , Article 13.Available at: http://scholarworks.uark.edu/jaas/vol22/iss1/1364¦iArkansas Academy of Science Proceedings, Vol. 22, 1968Microirradiation of LivingFungal Cells with a Laser 1John L. Robertson and Charles L. WilsonINTRODUCTIONThe introduction of the laser into biology has provided a toolwith which new questions can be asked, and possibly answered. Ithas proven to be a useful instrument for microsurgery because avery small area of the cell can be destroyed with little apparentdamage to the remaining portions. There is evidence that laserradiation can be used as a mutagen in much the same manner asx-ray or other types of radiation. This paper discusses the methodsinvolved in preparation of living fungal materials for irradiation witha laser beam and describes some responses of irradiated cells. Toour knowledge, experiments of this nature have not been performedpreviously with the fungal cell.MATERIALS AND METHODSMaterial to be irradiated was prepared by inoculating agar-dipped slides wth blocks of agar containing fungal mycelium. In-oculated slides were incubated at 24°C for 15 to 18 hours in amoist petri dish. Agar-dipped slides were prepared as follows: sterileslides, .96 to 1.06 mm in thickness, were dipped into hot Difco OakWilt agar in a sterile petri dish and allowed to become well covered.They were then held on end to drain off excess agar, leaving onlya thin film on the slide. After inoculation and incubation of thefungus the excess agar was scraped off the slide leaving a smallsquare of the agar with fungal hyphae. A cover slip, no. 0, was thenplaced on the slide and all edges of the coverslip were sealed withparaffin.A Leitz phase-contrast microscope with Heine condenser wasused in conjunction with a TRG bio-coupling model 153A lasersystem. Shots were fired through a Leitz 70x oil fluorite objective.The laser has a maximum power input of 230 joules and could befired once every minute with a pulse duration of 150 u sec. The laserwas aimed by use of a set of crosshairs in the ocular of the micro-scope. Because of the large size of its cells, Basidiobolus ranarunEidam was the fungus used in this study.RESULTSThe results of microirradiation are affected by the activityof the cell at the time of irradation. Ifthe cell is dividing, a muta-:Published with the approval of the Director of the Arkansas Agricultural Experiment Station.64Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 13Published by Arkansas Academy of Science, 196865|Laser Irradiation of Fungal Cellsgenie effect may be obtained. When a shot is directed into thechromatin at telophase, a portion of the chromatin is destroyed withthe resulting formation of a nucleus which is much reduced in size.If a shot is directed into the spindle during division, no cellwall subsequently forms, so that a cell with two nuclei is produced.When a nucleolus is irradiated at one end (Fig. 1) a darkenedarea appears at the focal point of the beam. After a period of about2 minutes the nucleolus can be seen to constrict at the point ofirradiation (Fig. 2). Approximately two hours after tihe initialshot, a portion of the nucleolus may separate from the main body(Fig. 3). The separated portion may fragment into small bodieswhich remain within the nuclear envelope. (Fig. 4).The elongation of a cell can be arrested by irradiation of thetip. This, however, must be done with care because it is quite possibleto burn a hole in the cell wall permitting the cytoplasm to escapethrough it. These holes generally range in size from 1 to 3 u (Fig. 5).DISCUSSIONTh<e amount of the cell damaged, and the type of damage doneby laser irradiation depends on several factors. The amount ofenergy input and the size of the aperture affects the size and in-tensity of the beam in the TRG system. Magnification also affectsthe size of the damaged area: the higher the magnification, thesmaller the damaged area. Ifthe microscope is out of focus, a muchlarger portion of the cell will be damaged. The depth at which thecell is embedded in the agar also determines how effective a shot willbe; this is one reason for the importance of the thin film of agaron the slide. Thin preparations are absolutely essential in order toobtain good results with laser irradiation through the microscope. Italso encourages the fungus to grow in one plane, thus allowing entirecells to be brought into focus at the same time.The reaction of the nucleolus when irradiated indicated thatthere is something which bounds its contents. Although no membranehas been found around the nucleolus with ultrastructure studies, theevidence presented here appears to indicate that one may be present.Such a membrane might not have the generally accepted unit mem-brane structure and therefore, would not be detectable by the methodsgenerally used in electron microscopy. A membrane that is highlyelastic and capable of repairing itself following breakage is suggestedby responses of the nucleolus to microirradiation.With laser radiation there is a possibility of increasing or re-ducing ploidy by selectively destroying the spindle or part of thenuclear material.65Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 13http://scholarworks.uark.edu/jaas/vol22/iss1/1366Arkansas Academy of Science ProceedingsLaser irradiation should provide a valuable tool for studies tobetter understand the fungal cell. The opportunity to selectivelydestroy parts of the cell should allow morpihpgenic and geneticstudies to be made that were hitherto impossible.DESCRIPTION OF FIGURESMicroirradiation of Basidiobolus Ranarum With LaserFig. 1. 650x Nucleolus shortly after irradiation. Arrow at pointof irradiation.Fig. 2. 650x. Constriction of same nucleolus (arrow) 2 minutesafter irradiation.Fig. 3 650x. The point of initial irradiation (R) at the far leftand constriction of nucleolus (c).Fig. 4. 1500x. Fragments (F) of nucleolus within the nuclearenvelope.Fig. 5. 1500x. Irradiated area at the tip.66Journal of the Arkansas Academy of Science, Vol. 22 [1968], Art. 13Published by Arkansas Academy of Science, 1968

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Microirradiation of Living Fungal Cells with a Laser

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