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TitleLight Microscopy: Methods and Protocols
Author
LanguageEnglish
File Size10.4 MB
Total Pages249
Table of Contents
                            Cover
Frontmatter
	Preface
	Contents
	Contributors
Section I Bright-Field Microscopy Applications
	1 Glycol Methacrylate Embedding for Improved Morphological, Morphometrical, and Immunohistochemical Investigations Under Light Microscopy: Testes as a Model
		1 Introduction
		2 Materials
		3 Methods
			3.1 Fixation and Storage
				3.1.1 Testes Fixation by Perfusion of Whole Body
				3.1.2 Fixation by Perfusion of Isolated Testis
				3.1.3 Testis Fixation by Immersion
				3.1.4 Comments
			3.2 Embedding
			3.3 Sectioning
			3.4 Staining
				3.4.1 For Morphological and Morphometrical Studies
				3.4.2 For Immunohistochemical Studies
			3.5 Photomicrography
		4 Notes
		References
	2 Histological Processing of Teeth and Periodontal Tissues for Light Microscopy Analysis
		1 Introduction
		2 Materials
			2.1 Cutting--Grinding Technique: Mineralized Samples
			2.2 Histological Processing for Demineralized Samples
		3 Methods
			3.1 Cutting--Grinding Technique: Mineralized Samples
			3.2 Routine Histological Processing of Demineralized Samples
				3.2.1 Samples Collection
				3.2.2 Sample Fixation and Demineralization
				3.2.3 Tissue Processing for Paraffin Embedding and Microtomy
				3.2.4 Staining
		4 Notes
		References
	3 Large Plant Samples: How to Process for GMA Embedding?
		1 Introduction
		2 Materials
		3 Methods
			3.1 Fixation
			3.2 Dehydration
			3.3 Pre-infiltration, Infiltration, and Embedding Treatments
			3.4 Microtomy and Staining
			3.5 Analysis
			3.6 Main Results and Discussion
		4 Notes
		References
	4 Image Cytometry: Nuclear and Chromosomal DNA Quantification
		1 Introduction
		2 Materials
			2.1 Calibration and System Setup
				2.1.1 Slide Preparation
				2.1.2 Feulgen Solution
				2.1.3 System Setup
			2.2 Preparation of Schiff's Reagent
			2.3 Slide Preparation for Ploidy Determination of Human Nuclear DNA by ICM
			2.4 Slide Preparation for Plant Nuclear DNA Quantification by ICM
			2.5 Slide Preparation for Plant Chromosomal DNA Quantification by ICM
		3 Methods
			3.1 Calibration and Setup of System
				3.1.1 Slide Preparation
				3.1.2 Feulgen Reaction (   see    Note 7)
				3.1.3 Microscopy and Digital System Setup
				3.1.4 Spatial Calibration -- From Pixel to Micrometer
				3.1.5 Density Calibration -- From OD to IOD
				3.1.6 Calibration Tests
			3.2 Slide Preparation for Ploidy Determination of Human Nuclear DNA by ICM
				3.2.1 Protocol Employing Air-Drying Technique
				3.2.2 Protocol Employing Cytocentrifuge Technique
			3.3 Slide Preparation for Plant Nuclear DNA Content Quantification by ICM
			3.4 Slide Preparation for Plant Chromosomal DNA Quantification by ICM (   see    Note 12)
			3.5 Image Cytometry
				3.5.1 Human Nuclear ICM
				3.5.2 Plant Nuclear ICM
				3.5.3 Plant Chromosomal ICM
		4 Notes
		References
	5 Histological Approaches to Study Tissue Parasitism During the Experimental    Trypanosoma cruzi    Infection
		1 Introduction
		2 Materials
			2.1 For Fixation
			2.2 For Processing and Embedding
			2.3 For Sectioning
			2.4 For Staining and Mounting
		3 Methods
			3.1 Fixation, Processing, and Embedding
			3.2 Sectioning
			3.3 Staining and Mounting
				3.3.1 Hematoxylin--Eosin
				3.3.2 Toluidine Blue-Basic Fuchsin
			3.4 Parasite Analysis and Quantification
		4 Notes
		References
	6 Intravital Microscopy to Study Leukocyte Recruitment In Vivo
		1 Introduction
		2 Materials
			2.1 Materials for Both Videomicroscopy
			2.2 Light Microscopy Set
			2.3 Epifluorescence Microscopy Set
		3 Methods
			3.1 Mouse Preparation for Intravital Microscopy
			3.2 Intravital Microscopy of Cremaster Muscle
			3.3 Intravital Microscopy of Knee Joint
			3.4 Evaluation of Leukocyte Recruitment
		4 Notes
		References
Section II Fluorescence Microscopy Applications
	7 Introduction to Fluorescence Microscopy
		1 A Brief History of Fluorescence Microscopy
			1.1 Invention of Fluorescence Microscopy
		2 What Is Fluorescence?
			2.1 The Chemical Basis of Fluorescence
		3 Properties of Fluorochromes
			3.1 Fluorescence Quantum Yield
			3.2 Quenching
			3.3 Photobleaching
			3.4 Molar Extinction
			3.5 Fluorescence Lifetime
			3.6 Stokes Shift
		4 Resolution in Fluorescence Microscopy
		5 The Construction of a Fluorescence Microscope
			5.1 Light Sources or Illuminators
				5.1.1 Operating Rules of Fluorescence Source Power Supplies
			5.2 Fluorescence Illuminator
				5.2.1 Registration Artifacts When Using Two or More Filters
				5.2.2 Orientation of the Fluorescence Filters in a Filter Cube
				5.2.3 Methods to Prevent Stray Light from Exiting the Filter Cube
			5.3 Objectives
				5.3.1 Adjusting the Correction Collar
		6 Phase Contrast and Fluorescence
		7 DIC and Fluorescence
			7.1 Condensers
		8 Trinocular Heads
		References
	8 Using the Fluorescent Styryl Dye FM1-43 to Visualize Synaptic Vesicles Exocytosis and Endocytosis in Motor Nerve Terminals
		1 Introduction
		2 Materials
		3 Methods
			3.1 Making Stock Solutions of FM1-43
			3.2 Monitoring Synaptic Vesicle Cycling in Frog Neuromuscular Junctions
				3.2.1 Staining Frog Neuromuscular Junctions Synaptic Vesicles with FM1-43
				3.2.2 Removing the Non-internalized FM1-43 from Frog Neuromuscular Junction Preparations
				3.2.3 Tissue Fixation
				3.2.4 Image Acquisition
				3.2.5 Destaining of Non-fixed Preparations of Frog Neuromuscular Junctions
				3.2.6 Image Analysis
			3.3 Monitoring Synaptic Vesicle Cycle in Mouse Neuromuscular Junctions
				3.3.1 Staining Mouse Neuromuscular Junctions Synaptic Vesicles with FM1-43
				3.3.2 Removing the Non-internalized FM1-43 in Preparations of Mouse Neuromuscular Junctions
				3.3.3 Tissue Fixation and Mounting of Slides
				3.3.4 Destaining of Non-fixed Mouse Neuromuscular Junctions
				3.3.5 Image Acquisition and Analysis
		4 Notes
		References
	9 Imaging Lipid Bodies Within Leukocytes with Different Light Microscopy Techniques
		1 Introduction
		2 Materials
			2.1 Osmium Staining
			2.2 Nile Red
			2.3 Oil Red O
			2.4 BODIPY
			2.5 1-Pyrenedodecanoic Acid
			2.6 Adipose Differentiation-Related Protein (ADRP, Adipophilin)
		3 Methods
			3.1 Sample Preparation onto Slides
			3.2 Osmium Staining
			3.3 Nile Red Staining
			3.4 Oil Red O Staining
			3.5 BODIPY Staining
			3.6 1-Pyrenedodecanoic Acid Staining
			3.7 ADRP Staining
			3.8 Lipid Body Analysis and Quantification
		4 Notes
		References
	10 EicosaCell _ An Immunofluorescent-Based Assay to Localize Newly Synthesized Eicosanoid Lipid Mediators at Intracellular Sites
		1 Introduction
		2 Materials
			2.1 Conventional EicosaCell
			2.2 Double-Labeling Purposes
		3 Methods
			3.1 EicosaCell with Cells in Suspension
			3.2 EicosaCell with Adherent Cells
			3.3 EicosaCell with Cells Embedded in a Gel Matrix
			3.4 Double-Labeling Procedures to Identify Eicosanoid-Synthesizing Intracellular Sites
				3.4.1 Nuclear Localization
				3.4.2 Phagosomal Localization
				3.4.3 Lipid Body Localization
		4 Notes
		5 Applications
		References
	11 Nestin-Driven Green Fluorescent Protein as an Imaging Marker for Nascent Blood Vessels in Mouse Models of Cancer
		1 Introduction
			1.1 Previous Models Used to Determine Angiogenesis
			1.2 Fluorescent Proteins to Image Angiogenesis
			1.3 Orthotopic Tumor Models Expressing Fluorescent Proteins to Visualize Tumor Angiogenesis
			1.4 Intravital Images of Angiogenesis of Orthotopic Pancreas Cancer
			1.5 Whole-Body Imaging of Angiogenesis in Orthotopic Breast Cancer
				1.5.1 Non-invasive Imaging of Tumor Blood Flow
			1.6 Skin Flaps Enable Ultra-high Resolution External Imaging of Tumor Angiogenesis
			1.7 Imaging of Nascent Angiogenesis Using Nestin-Driven GFP Transgenic Mice
				1.7.1 Dual-Color Imaging of Tumor Angiogenesis
			1.8 Nestin-Driven GFP Transgenic Nude Mice
			1.9 Imaging of Nascent Angiogenesis in Tumors in ND-GFP Nude Mice
			1.10 Rapid In Vivo/Ex Vivo Nascent Angiogenesis Assay
			1.11 A Brain- Metastatic Paralyzing Spinal Cord Glioma That Induces Angiogenesis and Neurogenesis
			1.12 Growth and Metastasis of Spinal Cord Glioma
			1.13 Efficacy of Chemotherapy on Spinal Cord Growth and Metastasis
			1.14 Efficacy of Chemotherapy on Hind-Limb Paralysis Due to Spinal Cord Glioma
			1.15 Angiogenesis and Neurogenesis of Spinal Cord Glioma
		2 Materials
			2.1 Reagents
			2.2 Equipment
		3 Methods
			3.1 Cell Injection to Establish an Experimental Metastasis Model
			3.2 Surgical Orthotopic Implantation to Establish a Spontaneous Metastasis Model (IND)
			3.3 Skin-Flap Windows
			3.4 Tumor Tissue Sampling
			3.5 Fluorescence Microscopy
			3.6 Methods for Color-Coded Imaging of Tumor Blood Vessels of Mice
				3.6.1 Microscopy
				3.6.2 Flashlight Imaging
				3.6.3 Light-Box Imaging
				3.6.4 Chamber Imaging
				3.6.5 Imaging with the Olympus IV-100 Laser Scanning Microscope System
				3.6.6 Imaging Using Spectral Separation
			3.7 Methods of Angiogenesis Analysis in GFP Models
				3.7.1 Fluorescence Contrast or Color-Coded Imaging
				3.7.2 Quantitative Analysis of Angiogenesis
			3.8 Evaluation of Anti-angiogenetic Agents
			3.9 Immunohistochemical Staining
		4 Notes
		References
	12 Imaging Calcium Sparks in Cardiac Myocytes
		1 Introduction
		2 Materials
		3 Methods
			3.1 Isolating Ventricular Myocytes from Adult Rat
			3.2 Fluo-4 AM Loading
			3.3 Ca
2+    Imaging in Ventricular Myocytes
			3.4 Recording Ca
2+    Transients in Ventricular Myocytes
			3.5 Recording Ca
2+    Sparks in Ventricular Myocytes
			3.6 Image Analyses
		4 Notes
		References
	13 Light Microscopy in Aquatic Ecology: Methods for Plankton Communities Studies
		1 Introduction
		2 Materials
			2.1 Virus
			2.2 Bacteria
			2.3 Phytoplankton
			2.4 Zooplankton
		3 Methods
			3.1 Virus Quantification
			3.2 Bacteria Enumeration
			3.3 Phytoplankton Enumeration
			3.4 Zooplankton
			3.5 Identification of Damaged Cells
		4 Notes
		References
	14 Fluorescence Immunohistochemistry in Combination with Differential Interference Contrast Microscopy for Studies of Semi-ultrathin Specimens of Epoxy Resin-Embedded Samples
		1 Introduction
		2 Materials
			2.1 Rat Tongues
			2.2 Preparation of Tissue
			2.3 Primary Antibodies
			2.4 Immunofluorescence Staining of K13 and CIII
			2.5 Light Microscopy (Axioplan; Carl Zeiss)
		3 Methods
			3.1 Preparation of Lingual Tissue
			3.2 Immunofluorescence Staining
			3.3 Light Microscopy
			3.4 Detection of Immunofluorescence of K13 in Rat Tongue on P7
			3.5 Detection of Immunofluorescence of K13 in Rat Tongue on P14
			3.6 Detection of Immunofluorescence of CIII in Rat Tongue on P7
			3.7 Detection of Immunofluorescence of CIII in Rat Tongue on P14
			3.8 Comparison Between Light Microscopy and Confocal Laser-Scanning Microscopy After Immuno-staining with Fluorescence-Labeled Antibodies
		4 Notes
		References
Subject Index
                        
Document Text Contents
Page 1







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METHODSIN
MOLECULARBIOLOGYTMSeriesEditor
JohnM.Walker
SchoolofLifeSciences
UniversityofHertfordshire
HatÞeld,Hertfordshire,AL109AB,UK
Forothertitlespublishedinthisseries,goto
www.springer.com/series/7651



Page 124




116Ghiran
Fig.7.8.Thebasiccon˚gurationofa˜uorescent˚lter.Partsareshownbeforeassembly
forabetterrepresentationoftherelationshipsbetweenvariouscomponents.Notethe
angledemission˚lter.Theorientationof˚ltersasshownby
arrows
ontheemissionand
excitation˚lterarenotuniversal.Seetextfordetails.
Fig.7.9.Representationofthethreesimple˚ltersusedfor˜uorescencemicroscopy
andtheirde˚ningparameters.Longpass(LP)andshortpass(SP)˚ltersarede˚ned
bytheircut-onandcut-offwavelengths.Thebandpass˚lters(Bandpass)arede˚nedby

theirpeaktransmission,centerwavelengths(CWL)andfull-widthhalfmaximum(FWHM)
(ImagecourtesyofChromaInc.).
transmission(Avg.%T),orforbandpass˚lters,thefull-widthhalf
maximum(FWHM),whichisameasureofthe˚lterslope(the

steeperthebetter).Combinationsofthesethree˚ltertypesare

foundinvirtuallyall˚ltercubes.
Figure7.10showsthediagrams
ofahigh-qualitysimple-bandDAPI˚lter.Thenovelmethods
employedtomanufacture˚ltershavereachedalmostthelowest

limitforauto-˜uorescenceoftheglassandthehighestlimitfor



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IntroductiontoFluorescenceMicroscopy117
Fig.7.10.Diagramshowingahigh-qualitysingle-bandDAPI˚lter.Notethealmostrect-
angularbandpassoftheexcitationandemission˚ltersthathavethe50%cut-onand
cut-offvaluesalmostidenticalwiththeblockingrange.Thedichromaticmirrorhasan

almostverticalslopeandcloseto0%transmittancefortheexcitationrangeandover
90%transmittancefortheemissionrange(DiagramcourtesyofSemrock,Inc.).
transmittance,barrierandslopeof˚lters.Notethealmostper-
fectwavelengthsselectionoftheinterference˚ltersthatensures

brightsignalsandblackbackground.
Excitation˚lters
:Inthepast,selectingthedesiredwave-
lengthswasrealizedbyusingcoloredsolutionsofspeci˚cdyes

thatwereplacedinglasscontainersinthelightpath.Colored
gelatinsheetsandcoloredglass˚lterslaterreplacedcontainers.
Theseapproachesforselectingtheexcitationwavelengthsworked

wellwithtrans-˜uorescencemethodswheresampleswereillumi-

natedthroughanimmersiondark˚eldcondenser,whichdueto
itsspeci˚cdesignpreventedmostofthehigh-energyexcitation
lighttoentertheobjectiveandallowedonlytheemitted˜uores-

cencetoformtheimage.Whilethese˚lterswereveryaffordable,
theysufferedfrompooropticalperformance,lowtransmittance
andmediocrewavelengthdiscrimination.Asigni˚cantproblem

wasthehighlevelofauto-˜uorescenceofdyesthatwereused
inthese˚lters,whichconsiderablydecreasedthesignal-to-noise
ratioofmostsamples.Inrecentyears,themajorityof˚ltersused

in˜uorescencemicroscopyareinterference˚lters.Themaintech-

nologiesusedtodayforconstructinganinterference˚lteruse
eithersoftorhardcoating.Softcoatingisbasedondepositionon
severaloptically˜atpiecesofglassgluedtogetherbyadhesiveof

multiplethin˚lmsofseveraldifferentdielectricsubstances(metal



Page 248




LIGHTMICROSCOPY
SubjectIndex
243Molarextinction
ormolarabsorption.............
99Œ100Mordantsolution...................................
12Morphometry..........................
3Œ17,20Œ21,73NND-GFP..................
186Œ187,189,193,196,201Seealso
Nestin-drivegreen˜uorescentprotein
orND-GFPNestin-drivegreen˜uorescentprotein
orND-GFP..........................
183Œ201Neurogenesis..................................
192Œ193Neuromuscularjunction...............
138,140Œ145,147Neuronalactivity..............................
137Œ138Neuronalcommunication...........................
138Neutraldensity.......
54,58,103,109Œ110,112Œ114,122Nilered.........................
151,154Œ156,158Œ160Nucleargenomesize.................................
52Numericaperture,(NA).......
54,94,100Œ102,115,123,126Œ130,209,230,232OOD..................................
52,57Œ60,62Œ64Seealso
Opticaldensity
Odontoblast.....................................
30,32OilredO.......................
151,154,156,158Œ159Opticaldensity......................................
52Orthotopically-growingtumor......................
185Orthotopicbreastcancer............................
185Orthotopicpancreascancer.........................
185PPapillaryepithelium...........................
234,237Paraf˚n......................
3,9Œ10,12,21,29Œ31,37,72Œ74,237embedding...............................
21,29,73Parasitenests..........................
70,73,75Œ76,78Parasitism.......................................
69Œ80PAS,
seeStain
Perfusion........
8Œ9,13,15,153,155,157,172,208Œ210Periodicacid-Schiffsolution.....................
3,6,13Periodontaltissues...............................
19Œ35Periodontium.........................
19Œ21,29,32,34Phasecontrast..............
113,129,131Œ132,167Œ168,171Œ172,212Phosphatebufferedsolution...........................
5Phosphorescence.............................
93,96Œ97Photobleaching...............
97,99,105,143,146,213Phytoplankton...................
216Œ218,220,222Œ225PlanckRychol™ssolution.............................
34Plankton
communities...............................
215Œ226Planktonicorganism...................
215,217,224PlantchromosomalICM...........
55Œ56,61Œ62,64Œ65Plantsample
DNAcontentinplant.....................
52,63Œ64largeplantsamples...........................
37Œ48plantnuclei...............................
53,55,62Planttissue...............................
37Œ38,46Œ47Plasticresin...............................
4,70,72Œ73Ploidydetermination.........................
55,59Œ61Polymerization
polymerizationofthesamplecore..............
44Œ45rapidpolymerization.............................
47unsatisfactorypolymerization.....................
47Propidiumiodide..................................
224Putt™seosin......................................
23,311-Pyrenedodecanoicacid(P-96).......
152,154,157Œ158QQuantumef˚ciency(QE).......................
98,105Seealso
Fluorescencequantumyield
Quenching................................
98Œ100,146RRed˜uorescentprotein
orRFP......................
184Refractiveindex.........
54,100,102,124Œ125,127Œ130,212,218Resinin˚ltration....................................
46Resinpolymerization......................
16,27,40,45Rhodamine..........................
82,84,87,89,158Ryanodinereceptor
orRyRs.........................
206SSchiff™sreagent...............................
54Œ56,66Sedgewick-Rafterchamber.........................
223Semi-thinsection....................................
4Semi-ultrathinsection................
229Œ231,234,238Silicone..........................
21,24Œ25,31,33,139Sphericalaberration..................
123Œ126,128Œ130Squashingtechnique.................................
66Stabilitytest.....................................
58Œ59Stain
BODIPY
R............151,154,156Œ160,166,170,173Œ174eosin.......
6,9,11Œ12,21,23,29,31,34,71,74Œ75,79,230erythrosine-orange-toluidine..............
5,9,11Œ12Feulgensolution..............................
53Œ54Gomori™strichrome.............
21,23,29,31Œ32,35Harris™hematoxilin.....................
6,23,31,71Nilered.....................
151,154Œ156,158Œ160OilredO....................
151,154,156,158Œ159PAS......................................
9,11,13Seealso
Periodicacid-Schiffsolution
Putt™seosin..................................
23,311-pyrenedodecanoicacid(P-96).........
152,154,157toluidineblue-basicfuchsin....................
74Œ76toluidineblue-borate....................
5,11Œ12,72toluidineblueO...........................
5,39,72Stokesshift...................................
100,120StyryldyeFM1-43............................
137Œ147Seealso
FM1-43Synapticvesicle...............................
137Œ147Synovialtissue...................................
85Œ86Syntheticrubberofwhitesilicone.....................
21TTeeth...........................................
19Œ35Temozolomide................................
192Œ193Testes,testis
acrossomalsystem.................................
3spermatogonia..............................
3Œ4,13Toluidineblue-basicfuchsin......................
74Œ76Toluidineblue-borate.......................
5,11Œ12,72ToluidineblueO..............................
5,39,72Tongue..................................
230,232Œ238Transcellularemigration..........................
82Œ83


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244LIGHTMICROSCOPY
SubjectIndex
Trypanosomacruzi
...............................69Œ80T.cruzi
nests....................................
77TypeIIIcollagen
orCIII...........................
230UUltravioletlight.....................................
94Undecalci˚edsofttissue.............................
20Undemineralizedsamples............................
20Uniformitytest.....................................
59VVacuumpump..........................
39,46,219,221disposablesyringe................................
46Virioplankton.................................
215,220WWavelength..........
83,94,97Œ100,103,105,108Œ109,113Œ117,119Œ120,124Œ125,126,173,184,197,199,208Workingdistance,(WD)..................
104,127,212XXenograft.........................................
184Xenonlamp..............................
105Œ107,109ZZooplankton.............................
216Œ217,219,223Œ224

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