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static Mat getpupil(Mat& frame,Point& centroids){
Mat pupilImg = Mat(frame.size(), CV_8UC1);
Mat grayImg = Mat(frame.size(), CV_8UC1);
Mat tframe = frame;
Mat resImg;
inRange(frame, Scalar(28,28,28), Scalar(75,75,75), pupilImg);
vector<vector<Point>> contours;
vector<Vec4i> hierarchy;
RNG rng(12345);
findContours(pupilImg, contours, hierarchy, CV_RETR_TREE, CV_CHAIN_APPROX_SIMPLE);
pupilImg.release();
pupilImg = frame.clone();
for (int i = 0; i < contours.size(); i++){
    Moments mom = moments(contours[i]);
    double area = mom.m00;
    if (area > 50){
        double pupilArea = area;
        double x = mom.m10/area;
        double y = mom.m01/area;
        vector<vector<Point>> pupils = contours;
        centroids = Point((int)x, (int)y);
        Scalar color = Scalar(0, 0, 0);
        drawContours(pupilImg, pupils,hierarchy[i][3], CV_RGB(0,0,0),CV_FILLED,8,hierarchy);
        break;
    }
}
return pupilImg;

}

Then we use Hough transform to find the Iris

static Mat getIris(Mat& frame,Point& centroid,int& rad){
Mat copyImg = frame.clone();
Mat Cf;
frame.copyTo(Cf);
Mat resImg = frame.clone();
Mat grayImg = Mat(frame.size(), CV_8UC1);
Mat mask = Mat(frame.size(),CV_8UC1);
Mat Storage = Mat(frame.cols, 1, CV_32FC3);
int x1, y1;
Canny(frame, grayImg, 5, 70, 3);
GaussianBlur(grayImg, grayImg, Size(7, 7), 0);
vector<Vec3f> circles = getCircles(grayImg);

for (int i = 0; i < circles.size(); i++){
    rad = cvRound(circles[i][2]);
    x1 = cvRound(circles[i][0]);
    y1 = cvRound(circles[i][1]);
    circle(mask, centroid, rad, CV_RGB(255, 255, 255), CV_FILLED);
    //circle(mask, Point(x1,y1), rad, CV_RGB(255, 255, 255), CV_FILLED);
    bitwise_not(mask, mask);
    subtract(frame,copyImg,resImg,mask);
    int x = centroid.x - rad;
    int y = centroid.y - rad;
    int w = rad * 2;
    int h = w;
           34    resImg = resImg(Rect(x,y,w,h));
    Mat cropImg = Mat(Size(w,h), CV_8UC1);
    resImg.copyTo(cropImg);
    resImg = resImg(Rect(0, 0, resImg.cols, resImg.rows));
    return cropImg;
}
return resImg;

}

Then i get error in line 34 above. OpencvError: Assertion failed (0 <=roi.x && 0 <= roi.width && ro.x + roi.width < m.cols && 0<=roi.height && roi.y + roi.height <= m.rows) in cv::Mat::Mat