Mikroskop Cahaya
Mikroskop Cahaya
SHORT COMMUNICATION
Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency of Indonesia
Jalan Lebak Bulus Raya No. 49, Kotak Pos 7043, Jakarta 12070, Indonesia
The presence of malarial pigment (haemozoin) due to Plasmodium infection is a common histopathological effect
in mouse liver. Previous research showed that by using a polarized light microscope, researchers were better able
to detect haemozoin in mouse liver histology section. Thus, the aim of this research was to compare the haemozoin
area observed by a conventional vs. simple polarized light microscope by using image processing analysis. A total of
40 images produced from both conventional light microscope and simple polarized light microscope were collected.
All images were analyzed using ImageJ 1.47 software to measure the haemozoin areas. Our results showed that non
birefringent haemozoin and birefringent haemozoin area was significantly different. This was because when using
conventional light microscope the brown area that contained images of non birefringent haemozoin images also
contained Kupffer cells which appeared as the same brown color as haemozoin. In contrast, haemozoin gave bright
effect and can be easily differentiated with Kupffer cells in the birefringent haemozoin images. This study concluded
that haemozoin detection in mouse liver histology using a simple polarized light microscope was more accurate
compared to that of conventional light microscope.
additional piece of polarizing material that had were made in 7 μm thickness using hematoxylin
been cut out of from a plastic polarizing lens or eosin (HE) stain and were obtained from the
a polarizing test strip. The researchers placed a Nuclear Biomedical Laboratory in the Center for
black polarizing sunglass on the top of light source Technology of Radiation Safety and Metrology,
of microscope and a piece of polarizing material National Nuclear Energy Agency of Indonesia. Mice
placed on the top of thick blood smear slide from were inoculated with the irradiated Plasmodium
a patient with severe malaria. This apparatus was berghei of an ANKA strain at a dose of 150 Gy and
able to can successfully identify haemozoin inside were kept for 24 days before proceeding to liver
the slide (Maude et al. 2009). Here in this study, we histological study.
adapted those techniques using a conventional light Image Acquisition. A Nikon Biophot microscope
microscope for polarized light microscope to detect attached to Nikon D3000 digital single lens reflects
haemozoin area in the mouse (Mus musculus sp.) (DSLR) camera system was used to capture the
liver section histology. The aim of this research was images of the blood smear slides. Images were
to compare the measurements of haemozoin area captured at a resolution of 1936 x 1296 pixel and
obtained using a conventional light microscope vs. saved as JPEG files. The first image was captured
a simple polarized light microscope by using image using the adapted polarized light microscope,
processing analysis. whereas the second image was captured using
the same setting but after removal of the linear
MATERIALS AND METHODS polarizing sheet and CPL filter. A total of 20
images were collected using simple polarized and
Polarized Light Microscope. A Nikon Biophot conventional light microscope.
microscope was used as a conventional light Image Analysis. A macro program was developed
microscope. The equipments required for adapting in ImageJ 1.47 for measuring the birefringent
Nikon Biophot to become a polarized light haemozoin area in mouse liver histology. The
microscope were a linear polarizing sheet (45 x 35 algorithm of macro program can be divided into the
mm) (Polar Pro) and commercial circular polarizing following several sequential steps (Figure 2). The
filter (CPL) 58 mm (Hoya Pro1) for Digital Single first step was to split the images channels into green,
Lens Reflex (DSLR) Camera. The CPL filter 58 mm blue and red channels. The second step was to invert
was placed on the top of the microscope light source the green channel image to detect the haemozoin
and the linear polarizing sheet was placed on the top area using thresholding methods. The last step
of liver histology slide. The slides were examined was to measure the haemozoin area and determine
under 40x objective lens with maximum brightness the outlined haemozoin area in the images. The
(Figure 1). The CPL filter was rotated in a clockwise haemozoin area in images captured using the
direction until it reached the position where the conventional light microscope was measured by
object appeared to be most dark. The difference using an ImageJ 1.47 plugin previously developed
between light and dark was approximately in 45° (unpublished data). To obtain the measurement of
of rotation. The angle at which the object appears the areas presented in micrometer (µm) the Set
darkest was noted (Kramer et al. 2001). Scale command in ImageJ must be employed.
Mouse (Mus musculus sp.) Liver Histology. First an image of a microscope stage micrometer
Mouse (Mus musculus sp.) liver histology slides under the same microscope magnification that
Figure 1. CPL filter 58 mm on top of the microscope light source (blue arrow) (left) and a linear polarizing sheet on the top of a
liver histology slide (blue arrow) (right).
50 RAMADHANI ET AL. HAYATI J Biosci
used to capture the haemozoin must be obtained. µm2, respectively. Scatter plots of the data points
Then with the straight line selection tool, we drew showed that the linear relation between birefringent
a line that corresponded to known distance in the and non birefringent haemozoin area was quite
microscope stage micrometer image. Subsequently, low (r =0.0785; Figure 3). Statistical analysis using
the known distance and unit of measurement was t-test showed that there was a significant different
entered in the Set scale command dialog. ImageJ between the area of birefringent and non birefringent
will automatically fill in the Distance in Pixels field haemozoin (P = 0.011).
based on the length of the line (Papadopolus et al.
2007). 14000
RESULTS 0
0 2000 4000 6000
Non birefringent haemozoin area (um²)
Birefringent and Non Birefringent Haemozoin
Figure 3. Scatter plots comparing birefringent and non birefringent
Area Measurement. The average area of haemozoin area.
birefringent and non birefringent haemozoin
observed in our samples (of 20 images) were A B
2843.47 ± 779.49 µm2 and 4101.151 ± 2053.75
A B
Combined image
Figure 2. ImageJ Macro flowchart for measure birefringent Figure 6. Haemozoin in unstained mouse liver histology (red
haemozoin area. circle) (Deroost et al. 2012).
Vol. 21, 2014 Short Communication���������
�������
51
Deroost K, Lays N, Noppen N, Martens E, Opdenakker G, Van Romagosa C, Menendez C, Ismail MR, Quintó L, Ferrer
den Steen PE. 2012. Improved methods for haemozoin B, Alonso PL, Ordi J. 2004. Polarisation microscopy
quantification in tissues yield organ-and parasite-specific increases the sensitivity of hemozoin and Plasmodium
information in malaria-infected mouse. Malar J 166:1-11. detection in the histological assessment of placental
Egan TJ. 2003. Haemozoin (malaria pigment): a unique malaria. Acta Trop 90:277-284. http://dx.doi.org/10.1016/j.
crystalline drug target. Targets 2:115-124. http://dx.doi. actatropica.2004.02.003
org/10.1016/S1477-3627(03)02310-9 Silva APC, Rodrigues SCO, Merlo FA, Paixao TA, Santos RL.
Frita R, Carapau D, Mota MM, Hanscheid T. 2012. In Vivo 2011. Acute and chronic histopathologic changes in wild
hemozoin kinetics after learance of Plasmodium berghei type or TLR-2-/-, TLR-4-/-, TLR-6-/-, TLR-9-/-, CD14-
infection in mouse. Malar Res Treat 2012:1-9. http:// /-, and MyD-88-/- mouse experimentally infected with
dx.doi.org/10.1155/2012/373086 Plasmodium chabaudi. Braz J Vet Pathol 4:5-12.
Hisaeda H, Yasutomo K, Himeno K. 2005. Malaria: immune Soniran OT, Idowu OA, Ajayi OL, Olubi C. 2012. Comparative
evasion by parasite. Int J Biochem Cell Biol 37:700-706. study on the effects of chloroquine and artesunate on
http://dx.doi.org/10.1016/j.biocel.2004.10.009 histopathological damages caused by Plasmodium berghei
Ismail MR, Ordi J, Menendez C, Ventura PJ, Aponte JJ, Kahigwa in four vital organs of infected albino mice. Malar Res
E, Hirt R, Cardesa A, Alonso PL. 2000. Placental pathology Treat 2012:1-7. http://dx.doi.org/10.1155/2012/960758
in malaria: an histological, immunohistochemical and Sullivan AD, Meshnick SR. 1996. Haemozoin: identification
quantitative study. Human Pathol 31:85-93. http://dx.doi. and quantification. Parasitol Today 12:161-163. http://
org/10.1016/S0046-8177(00)80203-8 dx.doi.org/10.1016/0169-4758(96)40001-1
Kramer B, Grobusch MP, Suttorp N, Neukammer J, Rinneberg Swedlow JR. 2012. Innovation in biological microscopy:
H. 2001. Relative frequency of malaria pigment- current status and future directions. Bioessays 34:333-340.
carrying monocytes of nonimmune and semi-immune http://dx.doi.org/10.1002/bies.201100168
patients from flow cytometric depolarized side scatter. Syaifudin M, Tetriana D, Darlina, Nurhayati S. 2011. The
Cytometry 45:133-140. http://dx.doi.org/10.1002/1097- feasibility of gamma irradiationfor developing malaria
0320(20011001)45:2<133::AID-CYTO1155>3.0.CO;2-K vaccine. JAI 37:91-101.
Lawrence C, Olson JA. 1986. Birefringent hemozoin identifies Wisse E, Braet F, Luo D, De Zanger R, Jans D, Crabbe E,
malaria. Am J Clin Pathol 86:360-363. Vermoesen AN. 1996. Structure and function of sinusoidal
Maude RJ, Buapetch W, Silamut K. 2009. Short Report: lining cells in the liver. Toxicol Pathol 24:100-111. http://
A Simplified, Low-cost method for polarized light dx.doi.org/10.1177/019262339602400114
microscopy. Am J Trop Med Hyg 81:782-783. http://dx.doi.
org/10.4269/ajtmh.2009.09-0383
Papadopolus F, Spinelli M, Valente S, Foroni L, Orrico C, Alviano
F. 2007. Common tasks in microscopic and ultrastructural
image analysis using ImageJ. Ultrastruct Pathol 31:401-
407. http://dx.doi.org/10.1080/01913120701719189
Copyright of HAYATI Journal of Biosciences is the property of Bogor Agricultural
University, Department of Biology and its content may not be copied or emailed to multiple
sites or posted to a listserv without the copyright holder's express written permission.
However, users may print, download, or email articles for individual use.